蛋白質のアミロイド線維形成における水の役割

• Project/Area Number: 15076206
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Osaka University
• Principal Investigator: 後藤 祐児 Osaka University, 蛋白質研究所, 教授 (40153770)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥45,900,000 (Direct Cost: ¥45,900,000); Fiscal Year 2007: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2006: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2005: ¥8,000,000 (Direct Cost: ¥8,000,000); Fiscal Year 2004: ¥11,800,000 (Direct Cost: ¥11,800,000); Fiscal Year 2003: ¥10,100,000 (Direct Cost: ¥10,100,000)
• Keywords: 生物物理 / ナノ材料 / 生体分子 / 蛋白質のフォールディング / 老化 / アミロイド線維 / 蛋白質の構造安定性 / 透析アミロイドーシス / 蛍光顕微鏡 / 水素 / 重水素交換 / β2ミクログロブリン / アミロイドβペプチド

Research Abstract

アミロイド線維は、蛋白質と水との相互作用の異常がもたらす構造状態である。アミロイド線維の構造安定性や形成機構を研究することは、蛋白質の構造や物性、水との相互作用に対する理解を深めるために重要である。透析アミロイドーシスに関わるβ2ミクログロブリンとアルツハイマー病に関わるAβペプチドを用いて、アミロイド線維の構造安定性やアミロイド線維形成の分子機構を研究して、以下の成果を得た。
1.線維形成のリアルタイム観察:既に、全反射蛍光顕微鏡とアミロイド線維に特異的に結合するチオフラビンTを用いて、線維の伸長反応を一線維レベルかつリアルタイムで観察する方法を開発し、β2ミクログロブリンやAβペプチドの線維形成を観察してきた。今年度は特に、自発重合によるAβアミロイド線維形成を観察し、極めて多様な線維の形成されることを明らかにした。これを基に、アミロイド超分子構造の多様性の重要性を提案した。
2.線維の配向と分光特性:アミロイド線維が流れによって配向し、特徴的な円二色性、直線二色性を示すことを発見した。特に直線二色性を基に、アミロイド線維の発色団の分子構造を推定できることを示した。
3.蛋白質の凝集によってアミロイド核形成が促進されることを示した。また、線維形成は水との相互作用において天然構造とは異なる大きな変化を伴なうこと、熱測定や体積変化によってこれを検出できることを示した。
全研究期間内では、蛋白質のフォールディングとアミロイド線維形成についての共通点と相違点を、明らかにすることができた。特に、水との相互作用において大きく異なる特徴を示した。アミロイド線維では露出した親水性基が水の広範な水素結合ネットワークを誘導している可能性がある。しかし、その分子論的な理解は未解決であり、今後の課題である。

Research Products

• [Journal Article] Heat-triggered conversion of protofibrils into mature amyloid fibrils of β2-microglobulin. (2007)
  • Author(s): Sasahara, K.
  • Journal Title: Biochemistry 46 Pages: 3286-3293
  • Peer Reviewed
• [Journal Article] Flow-induced alignment of amyloid protofilaments revealed by linear dichiroism. (2007)
  • Author(s): Adachi, R.
  • Journal Title: J. Biol. Chem. 282 Pages: 8978-8983
  • Peer Reviewed
• [Journal Article] Heat-induced efficient conversion of β2-microglobulin hen egg-whitelysozyme into amyloid fibrils (2007)
  • Author(s): Sasahara, K.
  • Journal Title: J. Mol Biol. 372 Pages: 981-991
  • Peer Reviewed
• [Journal Article] Visualization and classification of amyloid β supramolecular assemblies. (2007)
  • Author(s): Yagi, H
  • Journal Title: Biochemistry 46 Pages: 15009-15017
  • Peer Reviewed
• [Journal Article] Direct observation of amyloid growth monitored by total internal reflection fluorescence microscopy. (2006)
  • Author(s): Ban, T.
  • Journal Title: Methods Enzymol. 413 Pages: 91-102
• [Journal Article] Direct observation of amyloid fibril growth, propagation, and adaptation. (2006)
  • Author(s): Ban, T.
  • Journal Title: Acc. Chem. Res. 39 (9) Pages: 663-670
• [Journal Article] Conformation of amyloid fibrils of β_2-microglobulin probed by tryptophan mutagenesis. (2006)
  • Author(s): Kihara, M.
  • Journal Title: J. Biol. Chem. 281 (41) Pages: 31061-31069
• [Journal Article] Seeding-dependent propagation and adaptation of amyloid fibril conformation. (2006)
  • Author(s): Chatani, E.
  • Journal Title: J. Mol. Biol. 359 (4) Pages: 1086-1096
• [Journal Article] Real-time and single fibril observation of the formation of amyloid β spherulitic structures. (2006)
  • Author(s): Ban, T.
  • Journal Title: J. Biol. Chem. 281 (44) Pages: 33677-33683
• [Journal Article] 3D Structure of amyloid protofilaments of β2-microglobulin fragment probed by solid-state NMR. (2006)
  • Author(s): Iwata, K.
  • Journal Title: Proc. Natl. Acad. Sci. USA 103 (48) Pages: 18119-18124
• [Journal Article] Stereospecific amyloid fibril formation of a peptide fragment of β2-microglobulin. (2005)
  • Author(s): Wadai, H.
  • Journal Title: Biochemistry 44(1) Pages: 157-164
• [Journal Article] Critical balance of electrostatic and hydrophobic interactions is required for β2-microglobulin arnyloid fibril growth and stability. (2005)
  • Author(s): Raman, B.
  • Journal Title: Biochemistry 44(4) Pages: 1288-1299
• [Journal Article] Seeding-dependent maturation of β2-microglobulin amyloid fibrils at neutral pH. (2005)
  • Author(s): Kihara, M.
  • Journal Title: J.Biol.Chem. 280(1) Pages: 12012-12018
• [Journal Article] Metal ion-dependent effects of clioquinol on the fibril growth of an amyloid β peptide. (2005)
  • Author(s): Raman, B.
  • Journal Title: J.Biol.Chem. 280(16) Pages: 16157-16162
• [Journal Article] NMR characterization of refolding of β2-microglobulin trapped by prolyl cis-trans isomerization. (2005)
  • Author(s): Kameda, A.
  • Journal Title: J.Mol.Biol. 348(2) Pages: 383-397
• [Journal Article] Main-chain dominated amyloid structures demonstrated by the effect of high pressure. (2005)
  • Author(s): Chatani, E.
  • Journal Title: J.Mol.Biol. 352(4) Pages: 941-951
• [Journal Article] Kinetically controlled thermal response of β2-microglobulin amyloid fibrils. (2005)
  • Author(s): Sasahara, K.
  • Journal Title: J.Mol.Biol. 352(3) Pages: 700-711
• [Journal Article] αB-Crystallin, a small heat shock protein, prevents the amyloid fibril growth of an amyloid B-peptide and B2-microglobulin. (2005)
  • Author(s): Raman, B.
  • Journal Title: Biochem.J. 392(3) Pages: 573-581
• [Journal Article] Seeding-dependent propagation versus maturation of amyloid fibril conformation. (2005)
  • Author(s): Yamaguchi, K.
  • Journal Title: J.Mol.Biol. 352(4) Pages: 952-960
• [Journal Article] Ultrasonication-induced amyloid fibril formation of β2-microglobulin. (2005)
  • Author(s): Ohhashi, Y.
  • Journal Title: J.Biol.Chem. 280(38) Pages: 32843-32848
• [Journal Article] Structural model of the amyloid fibril formed by β2-microglobulin #21-31 fragment based on vibrational spectroscopy. (2005)
  • Author(s): Hiramatsu, H.
  • Journal Title: J.Amer Chem.Soc. 127(22) Pages: 7988-7989
• [Journal Article] Molecular interactions in the formation and deposition of β2-microglobulin-related amyloid fibrils. (2005)
  • Author(s): Naiki H.
  • Journal Title: Amyloid 12(1) Pages: 15-25
• [Journal Article] Kinetic analysis of the polymerization and depolymerization of β2-microglobulin-related amyloid fibrils in vitro. (2005)
  • Author(s): Yamamoto, S.
  • Journal Title: Biochim.Biophys.Acta (Special Issue) 1753 Pages: 64-75
• [Journal Article] Structural studies reveal that the diverse morphology of β2-microglobulin aggregates is a reflection of different molecular architectures. (2005)
  • Author(s): Kardos, J.
  • Journal Title: Biochim.Biochem.Acta (Special Issue) 1753 Pages: 108-120
• [Journal Article] アミロイド線維形成の原動力:エントロピー的排除容積効果 (2005)
  • Author(s): 木下正弘
  • Journal Title: 現代化学 4月号 Pages: 27-31
• [Journal Article] アミロイド線維形成機構-裏の世界の構築原理 (2005)
  • Author(s): 茶谷絵理
  • Journal Title: 実験医学 23 Pages: 174-180
• [Journal Article] Alcohol- and Salt-induced Partially Folded Intermediates. (2005)
  • Author(s): Hamada, D.
  • Journal Title: Protein Folding Handbook Vol.2 (Eds.J.Buchner and T.Kiefhaber) Pages: 884-916
• [Journal Article] Increase in the conformational flexibility of β2-microglobulin upon copper binding. (2004)
  • Author(s): Villanueva, James
  • Journal Title: Protein Science 13(3) Pages: 797-809
• [Journal Article] Oputimum amyloid fibril formation of peptide 20-41 at neutral pH suggests intrinsic amyloidogenic preference of β2-microglobulin. (2004)
  • Author(s): Ohhashi, Yukiko
  • Journal Title: J.Biol.Chem. 279(11) Pages: 10814-10821
• [Journal Article] Core and heterogeneity of β2-microglobulin amyloid fibrils as revealed by H/D exchange. (2004)
  • Author(s): Yamaguchi, Kei-ichi
  • Journal Title: J.Mol.Biol. 338(3) Pages: 559-571
• [Journal Article] Conformational stability of amyloid fibrils of β2-mictroglobulin probed by guanidine hydrochloride-induced unfolding. (2004)
  • Author(s): Narimoto, Takehiro
  • Journal Title: FEBS Letters 576(3) Pages: 313-319
• [Journal Article] Direct measurement of the thermodynamic parameters of amyloid formation by isothermal titration calorimetry. (2004)
  • Author(s): Kardos, Jozsef
  • Journal Title: J.Biol.Chem. 279(53) Pages: 55308-55314
• [Journal Article] Direct observation of Aβ amyloid fibril growth and inhibition. (2004)
  • Author(s): Ban, Tadato
  • Journal Title: J.Mol.Biol. 344(3) Pages: 757-767
• [Presentation] Direct observation of amyloid fibril growth and propagation (2007)
  • Author(s): 後藤祐児
  • Organizer: Conference on Structure and Dynamics in Soft Matter and Biomolecules
  • Place of Presentation: Trieste, Italy
• [Book] 「タンパク質の一生集中マスター」遠藤斗志也、森和俊、田口英樹編 (2007)
  • Author(s): 櫻井一正
  • Total Pages: 146
  • Publisher: 羊土社
• [Book] タンパク質の一生集中マスター(遠藤、森、田口編) (2007)
  • Author(s): 櫻井一正
  • Total Pages: 146
  • Publisher: 羊土社
• [Book] タンパク質科学「構造・物性・機能」 (2005)
  • Author(s): 後藤 祐児
  • Total Pages: 566
  • Publisher: 化学同人
• [Remarks]
  • URL: http://www.protein.osaka-u.ac.jp/
• [Publications] Fernandez, Ariel: "Structural defects and the diagnosis of amyloidogenic propensity"Proc.Natl.Acad.Sci.USA. 100. 6446-6451 (2003)
• [Publications] Gozu, Masayo: "Conformatinal dynamics of β2-microglobulin analyzed by reduction and reoxidation of the disulfide bond."J.Biochem.. 133. 731-736 (2003)
• [Publications] Fernandez, Ariel: "Protein folding : could hydrophobic collapse be coupled with hydrogen-bond formation?"FEBS Letters. 536. 187-192 (2003)
• [Publications] Ban, Tadato: "Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence."J.Biol.Chem.. 278. 16462-16465 (2003)
• [Publications] Hirota-Nakaoka, Nami: "Dissolution of β_2-microglobulin amyloid fibrils by dimethylsufloxide."J.Biochem.. 134. 159-164 (2003)
• [Publications] Chiba, Takeshi: "Amyloid fibril formation in the context of full-length protein : Effects of proline mutations on the amyloid fibril formation of β2-microglobulin."J.Biol.Chem.. 278. 47009-47015 (2003)