脂質膜環境でのカルシトニンおよびグルカゴンのアミロイド線維形成分子機構の解明

• Project Area: Dynamical ordering of biomolecular systems for creation of integrated functions
• Project/Area Number: 16H00756
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Yokohama National University
• Principal Investigator: 内藤 晶 横浜国立大学, 大学院工学研究院, 名誉教授 (80172245)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥5,720,000 (Direct Cost: ¥4,400,000、Indirect Cost: ¥1,320,000); Fiscal Year 2017: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2016: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: カルシトニン / グルカゴン / アミロイド線維 / 固体ＮＭＲ / 線維形成機構 / 中性リン脂質 / 固体NMR / 電子顕微鏡 / アミロイド線維中間体

Outline of Annual Research Achievements

カルシトニンは甲状腺から分泌され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るが、高濃度中性水溶液中でアミロイド様線維を形成して沈殿する性質を持っている。膵臓から分泌され血糖値を上昇させることに働くグルカゴンも、アミロイド様線維を形成しアポトーシスを誘発することが知られている。
これまで、カルシトニンに関しては酸性および中性条件でのカルシトニンのアミロイド線維形成機構について研究した結果以下の研究成果が得られた。１）カルシトニンはモノマーから核が形成される核形成反応(k1)を経て核にモノマーが吸着して線維が伸長する線維伸長反応(k2)の2段階の反応、すなわち2段階自己触媒反応機構で線維形成することが判明した。２）中性条件で線維は逆平行ｂ－シートを形成してｂ－ストランドに垂直な方向に成長する構造を明らかにした。また中央部のPhe16, Phe19はｂ－シート構造の安定化に寄与していることが判明した。３）２）の結果を踏まえて、PheをLeuに置換したhCT変異体では線維伸長反応速度が格段に遅くなる結果が得られた。Pheの相互作用が弱まり、線維の安定性が下がったためであることが判明した。４）今年度はクルクミンを添加してhCTの線維形成速度を算出したところ、核形成反応が格段に遅くなることが判明した。クルクミンがhCTモノマーと相互作用を形成するため、モノマー同士の会合能が下がったためであると結論した。
グルカゴンについては酸性水溶液条件で中性リン脂質DMPC存在下でアミロイド様線維形成速度を観測したところ、酸性水溶液中に比べて、核形成速度は促進され、線維形成速度は阻害されることが判明した。グルカゴンは膜存在下ではN-端のｂ－シートが形成されず、Ｃ－末端でのみｂ－シートを形成することから、膜中では線維の安定性が下がり、線維伸長速度が遅くなったと結論した。

Research Progress Status

29年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

29年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy (2018)
  • Author(s): Naito Akira、Matsumori Nobuaki、Ramamoorthy Ayyalusamy
  • Journal Title: Biochimica et Biophysica Acta (BBA) - General Subjects Volume: 1862 Issue: 2 Pages: 307-323
  • DOI: 10.1016/j.bbagen.2017.06.004
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Quantitative Analysis of Solid-State Homonuclear Correlation Spectra of Antiparallel beta-Sheet Alanine Tetramers (2018)
  • Author(s): A. Naito, K. Okushita, K. Nishimura, G. S. Boutis, A. Aoki, and T. Asakura
  • Journal Title: J. Phys. Chem. B Volume: 122 Issue: 10 Pages: 2715-2724
  • DOI: 10.1021/acs.jpcb.7b11126
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The role of d - allo -isoleucine in the deposition of the anti- Leishmania peptide bombinin H4 as revealed by 31 P solid-state NMR, VCD spectroscopy, and MD simulation (2018)
  • Author(s): Mijiddorj Batsaikhan、Kaneda Shiho、Sato Hisako、Kitahashi Yuki、Javkhlantugs Namsrai、Naito Akira、Ueda Kazuyoshi、Kawamura Izuru
  • Journal Title: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Volume: 1866 Issue: 7 Pages: 789-798
  • DOI: 10.1016/j.bbapap.2018.01.005
  • NAID: 120006649291
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] 生体幕内でのペプチドの挙動の分子動力学シミュレーション (2018)
  • Author(s): 上田一義、内藤 晶、川村 出
  • Journal Title: 酵素工学ニュース Volume: 79 Pages: 27-39
  • Peer Reviewed
• [Journal Article] 固体高分解能NMRスペクトルに現れる異方性相互作用-Narrrow is beautifull- (2018)
  • Author(s): 内藤 晶
  • Journal Title: 分光研究 Volume: 63 Pages: 13-15
  • Peer Reviewed
• [Journal Article] Retinal configuration of ppR intermediates revealed by photo-irradiation solid-state NMR and DFT (2018)
  • Author(s): Makino Yoshiteru, Kawamura Izuru, Okitsu Takashi, Wada Akimori, Kamo Naoki, Sudo Yuki, Ueda Kazuyoshi, Naito Akira
  • Journal Title: Biophysical Journal Volume: in press
  • NAID: 120006698118
  • Peer Reviewed
• [Journal Article] Dynamic structure and orientation of melittin bound to acidic lipid bilayers, as revealed by solid-state NMR and molecular dynamic simulation (2017)
  • Author(s): K. Norisada, N. Javkhlantugs, D. Mishima, I. Kawamura, H. Saito, K. Ueda, A. Naito
  • Journal Title: J. Phys. Chem. B. Volume: 121 Issue: 8 Pages: 1802-1811
  • DOI: 10.1021/acs.jpcb.6b11207
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Packing arrangement of 13C selectively labeled sequence model peptides of Samia cynthia ricini silk fibroin fibers studied by solid-state NMR (2017)
  • Author(s): Asakura Tetsuo、Miyazawa Kenta、Tasei Yugo、Kametani Shunsuke、Nakazawa Yasumoto、Aoki Akihiro、Naito Akira
  • Journal Title: Physical Chemistry Chemical Physics Volume: 19 Issue: 20 Pages: 13379-13386
  • DOI: 10.1039/c7cp01199g
  • Peer Reviewed
• [Journal Article] Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations (2017)
  • Author(s): Asakura Tetsuo、Nishimura Akio、Kametani Shunsuke、Kawanishi Shuto、Aoki Akihiro、Suzuki Furitsu、Kaji Hironori、Naito Akira
  • Journal Title: Biomacromolecules Volume: 18 Issue: 6 Pages: 1965-1974
  • DOI: 10.1021/acs.biomac.7b00441
  • Peer Reviewed
• [Journal Article] Growth-incompetent monomers of human calcitonin lead to a noncanonical direct relationship between peptide concentration and aggregation lag time (2017)
  • Author(s): Kamgar-Parsi Kian、Hong Liu、Naito Akira、Brooks Charles L.、Ramamoorthy Ayyalusamy
  • Journal Title: Journal of Biological Chemistry Volume: 292 Issue: 36 Pages: 14963-14976
  • DOI: 10.1074/jbc.m117.791236
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Packing Arrangements and Intersheet Interaction of Alanine Oligopeptides As Revealed by Relaxation Parameters Obtained from High-Resolution 13C Solid-State NMR (2017)
  • Author(s): Naito Akira、Tasei Yugo、Nishimura Akio、Asakura Tetsuo
  • Journal Title: The Journal of Physical Chemistry B Volume: 121 Issue: 38 Pages: 8946-8955
  • DOI: 10.1021/acs.jpcb.7b07068
  • Peer Reviewed
• [Journal Article] Packing arrangement of 13C selectively labeled sequence model peptides of samia Cynthia ricini silk fibroin fiber studied by solid-state NMR, (2017)
  • Author(s): T. Asakura, K. Miyazawa, Y. Tasei, S. Kametani, Y. Nakazawa, A. Aoki, A. Naito
  • Journal Title: Phys. Chem. Chem. Phys. Volume: in press
  • Peer Reviewed
• [Journal Article] Parallel b-sheet structure of alanine tetrapeptide in the solid state as studied by solid-state NMR spectroscopy (2016)
  • Author(s): T. Asakura, K. Horiguchi, A. Aoki, Y. Tasei, A. Naito
  • Journal Title: J. Phys. Chem B. Volume: 120 Issue: 34 Pages: 8912-8941
  • DOI: 10.1021/acs.jpcb.6b06292
  • Peer Reviewed
• [Journal Article] Structural biology of calcitonin: from aqueous therapeutic properties to amyloid aggregation (2016)
  • Author(s): K Kamgar-Parsi, J. Tolchard, B. Habenstein, A. Loquest, A. Naito, A. Ramamoorthy
  • Journal Title: Isr. J. Chem. Volume: in press Issue: 7-8 Pages: 634-650
  • DOI: 10.1002/ijch.201600096
  • Peer Reviewed / Int'l Joint Research
• [Presentation] Dynamic membrane bound structure of melittin and alamethicin as revealed by solid-stae NMR and MD simulation (2018)
  • Author(s): Naito, T. Nagao, K. Norisada, N. Javkhlantugs, D. Mishima, I. Kawamura, K. Ueda
  • Organizer: Biophysical Society 62nd Annual Meeting, San Francisco, USA
  • Int'l Joint Research
• [Presentation] Microwave irradiation solid-state NMR spectroscopy to reveal microwave heating and energyflow mechanism of liquid crystals (2017)
  • Author(s): A. Naito
  • Organizer: The 7th Asia Pacific NMR Symposium, Bangalore (India)
  • Int'l Joint Research / Invited
• [Presentation] マイクロ波および光照射固体ＮＭＲの開発と液晶および光受容膜タンパク質への応用 (2017)
  • Author(s): 内藤 晶
  • Organizer: 第61回 固体ＮＭＲ・材料フォーラム、理化学研究所、横浜
  • Invited
• [Presentation] 13C固体ＮＭＲ（ＤＡＲＲ）を用いた13C選択標識エリ蚕絹モデルペプチドのパッキング様式の解析 (2017)
  • Author(s): 内藤 晶
  • Organizer: 第66回高分子学会年次大会、幕張メッセ
• [Presentation] Photo-intermediates in the photo-reaction pathways of bacteriorhodopsin as revealed by in-situ photo-irradiation solid-state NMR (2017)
  • Author(s): K. Oshima, Y. Otani, A. Shigeta, Y. Makino, I. Kawamura, T. Okitsu, A. Wada, S. Tuzi, T. Iwasa, A. Naito
  • Organizer: ISMAR2017, Quebrc, Canada
  • Int'l Joint Research
• [Presentation] Dynamic structure and orientation of Melittin and alamethicin as revealed by solid-state NMR and MD simulation (2017)
  • Author(s): A. Naito, T. Nagao, K. Norisada, N. Javkhlantugs, D. Mishima, D. Ishioka, K. Yokota, J. Wong, I. Kawamura, H. Saito, K. Ueda
  • Organizer: ISMAR2017, Quebrc, Canada
  • Int'l Joint Research
• [Presentation] Pressure and photo induced structural changes of photoreceptor proteins as revealed by solid-state NMR (2017)
  • Author(s): A. Naito
  • Organizer: IMBP2017, Kyoto (Japan)
  • Int'l Joint Research / Invited
• [Presentation] Membrane binding structure of Bombinin H2 and H4 peptides in leishmanial mimetic membrane as studied by solid-state NMR and MD simulation (2017)
  • Author(s): M. Batsaikhan, S. Kaneda, N. Javkhalantugs, K. Ueda, H. Sato, A. Naito, I. Kawamura
  • Organizer: 第55回生物物理学会年会、熊本
• [Presentation] In situ光照射固体ＮＭＲによるバクテリオロドプシンとその変異体に生成する光反応中間体の定常捕捉 (2017)
  • Author(s): 内藤 晶、大島恭介、大谷優人、重田安里寿、槇野義輝、川村 出、沖津貴志、和田昭盛、辻 暁、岩佐達郎
  • Organizer: 第55回生物物理学会年会、熊本
• [Presentation] 13C NMR 化学シフト計算によるセンサリーロドプシンの光中間体におけるレチナール構造の解析 (2017)
  • Author(s): Y. Makino, I. Kawamura, T. Okitsu, A. Wada, Y. Sudo, N. Kamo, A. Naito, K. Ueda
  • Organizer: 第55回生物物理学会年会、熊本
• [Presentation] Microwave heating mechanism of liquid crystals as revealed by microwave irradiation NMR (2017)
  • Author(s): A. Naito
  • Organizer: Magnetism and Magnetic Materials, London (UK)
  • Int'l Joint Research / Invited
• [Presentation] Photoirradiation and microwave irradiation NMR spectroscopy. Application to photoreceptor retinal proteins and liquid crystals. (2017)
  • Author(s): A. Naito
  • Organizer: Taiwan-Japan Biomedical Symposium on Magnetic Resonance, Tainan (Taiwan)
  • Int'l Joint Research / Invited
• [Presentation] センサリーロドプシンのレチナール構造変化に対する13C NMR化学シフト値計算 (2017)
  • Author(s): 槇野義輝、川村 出、沖津貴志、和田昭盛、須藤雄気、加茂直樹、内藤 晶、上田一義
  • Organizer: 第５６回ＮＭＲ討論会(2017)、首都大学東京
• [Presentation] 固体ＮＭＲによるPhe-Pheモチーフをもつペプチドの自己組織化集合体の分子構造の解析 (2017)
  • Author(s): 尾崎夢実、Batsaikhan, Mijiddori, 佐藤 久子、内藤 晶、川村 出
  • Organizer: 第５６回ＮＭＲ討論会(2017)、首都大学東京
• [Presentation] マイクロ波照射ＮＭＲ分光法による液晶分子のマイクロ波加熱分子機構の解明 (2017)
  • Author(s): 内藤 晶、田制侑悟、槇野義輝、藤戸輝明、川村 出
  • Organizer: 第５６回ＮＭＲ討論会(2017)、首都大学東京
• [Presentation] Secondary structural change of glucagon during fibril formation process with DMPC lipid bilayers as revealed by 13C solid-state NMR (2016)
  • Author(s): K. Haya, I. Kawamura, A. Naito
  • Organizer: 第54回生物物理学会年会
  • Place of Presentation: つくば国際会議場
  • Year and Date: 2016-11-25
• [Presentation] Structure of Tyr185 in dark-adapted bacteriorhodopsin as studied by solid-state NMR (2016)
  • Author(s): Y. Otani, A. Shigeta, Y. Kebukawa, K. Kobayashi, T. Okitsu, A. Wada, S. Tuzi, A. Naito, I. Kawamura
  • Organizer: 第54回生物物理学会年会
  • Place of Presentation: つくば国際会議場
  • Year and Date: 2016-11-25
• [Presentation] Specific interaction of cationic antimicrobial peptide bombinin H2 and H4 with Leishmania protozoa mimetic membrane (2016)
  • Author(s): S. Kaneda, A. Naito, I. Kawamura
  • Organizer: 第54回生物物理学会年会
  • Place of Presentation: つくば国際会議場
  • Year and Date: 2016-11-25
• [Presentation] Structure and orientation of antimicrobial peptides alamethicin and melittin in membrane revealed by solid state NMR and MD simulation (2016)
  • Author(s): A. Naito, T. Nagao, D. Mishima, N. Javkhlantugs, J. Wang, D. Ishioka, K. Yokota, K. Norisada, I. Kawamura, K. Ueda
  • Organizer: 第54回生物物理学会年会
  • Place of Presentation: つくば国際会議場
  • Year and Date: 2016-11-25
• [Presentation] Characterization of photo intermediates in sensory rhodopsin II as revealed by in-situ photoirradiation solid-state NMR (2016)
  • Author(s): Y. Makino, I. Kawamura, T. Okitsu, A. Wada, Y. Sudo, N. Kamo, A. Naito, K. Ueda
  • Organizer: 第54回生物物理学会年会
  • Place of Presentation: つくば国際会議場
  • Year and Date: 2016-11-25
• [Presentation] High resolution solid-state NMR spectroscopy. Development and application to structural biochemistry (2016)
  • Author(s): A. Naito
  • Organizer: 第55回ＮＭＲ討論会
  • Place of Presentation: 広島
  • Year and Date: 2016-11-16
  • Invited
• [Presentation] １３C固体NMRを用いたDMPC脂質二重膜存在下におけるペプチドホルモングルカゴンの線維構造の解析 (2016)
  • Author(s): 羽矢和未、川村 出、内藤 晶
  • Organizer: 第55回ＮＭＲ討論会
  • Place of Presentation: 広島
  • Year and Date: 2016-11-16
• [Presentation] 光照射固体NMRによるセンサリーロドプシンIIの光反応過程における光中間体構造の解明 (2016)
  • Author(s): 槙野義輝、川村 出、沖津貴志、和田昭盛、須藤勇雄気、加茂直樹、内藤 晶、上田一義
  • Organizer: 第55回ＮＭＲ討論会
  • Place of Presentation: 広島
  • Year and Date: 2016-11-16
• [Presentation] In-situ光照射固体NMRによるバクテリオロドプシンD96N変異体の光反応過程でのレチナールおよびタンパクシ質構造変化の解析 (2016)
  • 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55回ＮＭＲ討論会
  • Place of Presentation: 広島
  • Year and Date: 2016-11-16
• [Presentation] ３１P固体NMRによる抗菌ペプチドボンビニンH2およびH4の細菌模倣膜に対する作用の解析 (2016)
  • Author(s): 金田志穂、北橋由紀、内藤 晶、川村 出
  • Organizer: 第55回ＮＭＲ討論会
  • Place of Presentation: 広島
  • Year and Date: 2016-11-16
• [Presentation] Photoreaction pathways of bacteriorhodopsin and its mutants as revealed by in-situ photo-irradiation solid-state NMR (2016)
  • Author(s): A. Naito, Y. Makino, K. Oshima, Y. Otani, A. Shigeta, I. Kawamura, T. Okitsu, A. Wada, S. Tuzi,, T. Iwasa
  • Organizer: ICRP2016
  • Place of Presentation: Potsdam, Germany
  • Year and Date: 2016-10-02
  • Int'l Joint Research
• [Presentation] Structure and orientation of antibiotic peptide alamethicin in membrane environment as revealed by chemical shift oscillation analysis of solid state NMR and MD simulation (2016)
  • Author(s): T. Nagao, D. Mishima, N. Javkhalantugs, J. Wang, D. Ishioka, K. Yokota, K. Norisada, I. Kawamura, K. Ueda, A. Naito
  • Organizer: ICBRBS2016
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-08-21
  • Int'l Joint Research / Invited
• [Presentation] Analysis of interaction of antimicrobial peptides bombinin H2 and H4 with the mimetic bacterial membrane by 31P solid-state NMR (2016)
  • Author(s): S. Kaneda, Y. Kitahashi, I. Kawamura, A. Naito
  • Organizer: ICBRBS2016
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-08-21
  • Int'l Joint Research
• [Presentation] Structural change of Tyr185 in the vicinity of the retinal chromophore in bacteriorhodopsin by 13C solid-state NMR (2016)
  • Author(s): Y. Otani, A. Shigeta, Y. Kebukawa, K. Kopbayashi, T. Okitsu, A. Wada, S. Tuzi, A. Naito, I. Kawamura
  • Organizer: ICBRBS2016
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-08-21
  • Int'l Joint Research
• [Presentation] Structural changes of retinal in sensory rhodopsin II as revealed by in situ photoirradiation solid-state NMR (2016)
  • Author(s): Y. Makino, I. Kawamura, T. Okitsu, A. Wada, S. Sudo, N. Kamo, A. Naito, I. Kawamura, K. Ueda
  • Organizer: ICBRBS2016
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-08-21
  • Int'l Joint Research
• [Presentation] 固体NMRとMDシミュレーションによるリン脂質二分子膜に結合した抗生物質ペプチドアラメチシンの構造と配向の解明 (2016)
  • Author(s): 永尾隆、三島大輔、ジャブカランタグス ナムズライ、王 クン、石岡大輔、横田恭宣、則定和志、川村 出、上田一義、内藤 晶
  • Organizer: 日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 2016-06-07
• [Book] Photoirradiation and microwave irradiation NMR. Experimental Approaches of NMR Spectroscopy. Methodology and Applicatioin to Life Science and Material Science (2018)
  • Author(s): A. Naito, Y. Makino, Y. Tasei, I. Kawamura
  • Total Pages: 36
  • Publisher: Springer
• [Book] Structure determination of membrane peptides and proteins by solid-state NMR. Experimental Approaches of NMR Spectroscopy. Methodology and Applicatioin to Life Science and Material Science (2018)
  • Author(s): I. Kawamura, K. Norisada, A. Naito
  • Total Pages: 44
  • Publisher: Springer
• [Book] 生命科学のための物理化学15講 (2018)
  • Author(s): 功刀 滋、内藤 晶
  • Total Pages: 256
  • Publisher: 講談社
  • ISBN: 9784061538986
• [Book] In-situ photo irradiation solid-state NMR spectroscopy applied to retinal-binding membrane proteins. Modern Magnetic Resonance (2017)
  • Author(s): A. Naito, Y. Makino, I. Kawamura
  • Total Pages: 22
  • Publisher: Springer International Publishing
• [Book] In-situ photo irradiation solid-state NMR spectroscopy applied to retinal-binding membrane protein, Modern Magnetic Resonance (2017)
  • Author(s): A. Naito, Y. Makino, I. Kawamura
  • Publisher: Springer
• [Book] Photoirradiation and microwave irradiation NMR spectroscopy. Experimental approaches of NMR spectroscopy. Methodology and Application of Life Science and Material Science (2017)
  • Author(s): A. Naito, Y. Makino, Y. Tasei, I. Kawamura
  • Publisher: Springer
• [Book] 固体高分解能NMR(Narrow is beautiful)の構造生命化学研究への展開 NMR (Bulletin of the nuclear magnetic society of japan) (2016)
  • Author(s): 内藤 晶
  • Total Pages: 3
  • Publisher: 日本核磁気共鳴学会