Development of high precision method to detect amyloid formation for Alzheimer's disease

• Project/Area Number: 17550085
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Analytical chemistry
• Research Institution: Tokyo University of Pharmacy and Life Science
• Principal Investigator: TASHIRO Sakurako Tokyo University of Pharmacy and Life Science, School of Pharmacy, Lecturer (40328555)
• Co-Investigator(Kenkyū-buntansha): KOJIMA Masaki Iwate Medical University, School of Pharmacy, Associate Professor (90277252) ; TASHIRO Mitsuru Meisei University, Faculty of Science and Technology, Associate Professor (40315750)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥3,510,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥210,000); Fiscal Year 2007: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
• Keywords: amyloid fibrils / Alzheimer's disease / familial Parkinson's disease / α-synuclein / intermediate / アミロイド / NACP

Research Abstract

NAC (Non-Aβ-Component of amyloid) was identified as one of the component in amyloid fibril for Alzheimer's disease during the purification process of amyloid fibrils. Later its precursor protein, α-synuclein was cloned. Moreover, the point mutations of this precursor protein (A30P, A53T, E46K) were identified in the confirmed familial cases of autosomal-dominantly inherited, early-onset Parkinson's disease(PD). Now α-synuclein is used as an marker protein for Lewy bodies in PD patient in order to diagnose the disease.
In this study, we have attempted to examine the structural changes during the early stages of amyloid formation and also the effects of point mutations mentioned above using thioflavin T fluorescence, CD, NMR, small angle X-ray scattering (SAXS), and electron microscopy in order to elucidate the fibrillation mechanisms at an atomic or molecular level.
SVD analysis of CD and SAXS data obtained during amyloid formation of α-synuclein has revealed the possible existence of intermediate during the early stage of amyloid formation. Also the analysis of 1H-15N HSQC spectra at the early stage of fibrillation indicated the decrease in peak intensities corresponding to KTKEGV motif. As the decrease in peak intensities could be correlated to increase in molecular weight, KTKEGV regions might be involved in the early stage of fibril formations. Furthermore, the detailed SVD analysis of SAXS data showed possibility of the early stage intermediate being heptamer.

Research Products

• [Journal Article] Characterization of Fibrillation Process of α-Synuclein at the initial stage (2008)
  • Author(s): Tashiro M, Kojima M, Kihara, H., Kasai, K., Kamiyoshihara T, Ueda K, Shimotakahara S.
  • Journal Title: Biochem Biophys Res Commun 369 Pages: 910-914
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Review: Protein Sample Preparation for Structural Biology (2008)
  • Author(s): Shimotakahara S.
  • Journal Title: Bunseki 4 Pages: 178-182
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Characterization of Fibrillation Process of α-Synuclein at the initial stage (2008)
  • Author(s): Tashiro, M., Kojima, M., Kihara, H., Kasai, K., Kamiyoshihara, T., Ueda, K., Shimotakahara, S
  • Journal Title: Biochem Biophys Res Commun 369 Pages: 910-914
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Review : Protein Sample Preparation for Structural Biology (2008)
  • Author(s): Shimotakahara (Tashiro), S
  • Journal Title: Bunseki 4 Pages: 178-182
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Characterization of Fibrillation Process of a-Synuclein at the initial stage (2008)
  • Author(s): Tashiro M, Kojima M, Kihara, H., Kasai, K., Kamiyoshihara T, Ueda K, Shimotakahara S.
  • Journal Title: Biochem Biophys Res Commun (in press)
  • Peer Reviewed
• [Journal Article] 構造生物学におけるタンパク質の調製方法 (2008)
  • Author(s): Tashiro S.
  • Journal Title: 「ぶんせき」-進歩総説 (in press)
• [Journal Article] Obseration of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis (2007)
  • Author(s): Kamiyoshihara T, Kojima M, Ueda K, Tashiro M, Shimotakahara S.
  • Journal Title: Biochem Biophys Res Commun 355 Pages: 398-403
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC_η 1 (2007)
  • Author(s): Imai, Toru; Kasai, Kouki; Kurita, Junichi; Fukami, Kiyoko; Tashiro, Mitsuru; Shimotakahara, Sakurako
  • Journal Title: Protein Expression and Purification 56 Pages: 247-252
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Collective solvent flows around a protein investigated by molecular dynamics simulation (2007)
  • Author(s): Umezawa K, Higo J, Shimotakahara S, Shindo H.
  • Journal Title: J Chem Phys 127 Pages: 45101-45107
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis (2007)
  • Author(s): Kamiyoshihara, T., Kojima, M., Ueda, K., Tashiro, M., Shimotakahara, S
  • Journal Title: Biochem Biophys Res Commun 355(2) Pages: 398-403
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression and characterization of a pleckstrin homology domain in phospholipase C, PLCη 1 (2007)
  • Author(s): Imai, Toru ; Kasai, Kouki ; Kurita, Junichi ; Fukami, Kiyoko ; Tashiro, Mitsuru ; Shimotakahara, Sakurako
  • Journal Title: Protein Expression and Purification 56(2) Pages: 247-252
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Collective solvent flows around a protein investigated by molecular dynamics simulation (2007)
  • Author(s): Umezawa, K., Higo, J., Shimotakahara, S., Shindo, H
  • Journal Title: J Chem Phys 127(4) Pages: 45101-45101
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Observation of multiple intermediates in alpha-synuclein fibril formation by singular value decomposition analysis (2007)
  • Author(s): Kamiyoshihara T, Kojima M, Ueda K, Tashiro M, Shimotakahara S.
  • Journal Title: Biochem Biophys Res Commun 355 Pages: 398-403
  • Peer Reviewed
• [Journal Article] Expression and characterization of a pleckstrin homology domain in phospholipase C, PLC-η 1. (2007)
  • Author(s): Imai, Toru; Kasai, Kouki; Kurita, Junichi; Fukami, Kiyoko; Tashiro, Mitsuru; Shimotakahara, Sakurako
  • Journal Title: Protein Expression and Purification 56 Pages: 247-252
  • Peer Reviewed
• [Journal Article] Collective solvent flows around a protein investigated by molecular dynamics simulation. (2007)
  • Author(s): Umezawa K, Higo J, Shimotakahara S, Shindo H.
  • Journal Title: J Chem Phys 127 Pages: 45101-7
  • Peer Reviewed
• [Journal Article] Observation of Water Molecules Bound to a Protein Using Cold-Spray Ionization Mass Spectrometry (2005)
  • Author(s): Y.Sei, S.Shimotakahara, J.Ishii, H.Shindo, H.Seki, K.Yamaguchi, M.Tashiro
  • Journal Title: Anal.Sci. 21 Pages: 449-451
  • NAID: 10015513763
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Observation of water molecules bound to a protein using cold-spray ionization mass spectrometry (2005)
  • Author(s): Sei, Y., Shimotakahara, S., Ishii, J., Shindo, H., Seki, H., Yamaguchi, K., Tashiro, M
  • Journal Title: Anal. Sci 21 Pages: 449-451
  • NAID: 10015513763
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Observation of water molecules bound to a protein using cold-spray ionization mass spectrometry (2005)
  • Author(s): Sei, Y
  • Journal Title: Analytical Science 21 Pages: 449-451
• [Journal Article] Application of NMR screening techniques for observing ligand binding with a protein receptor. (2005)
  • Author(s): Shimotakahara, S
  • Journal Title: Magnetic Resonance Chemistry 43 Pages: 69-72
• [Presentation] 蛋白質周辺における水分子の流れ、分子動力学計算による解析 (2007)
  • Author(s): 梅澤 公二, 肥後 順一, 山岸 明彦, 田代(下高原) 桜子, 神藤 平三郎
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-21
• [Presentation] uilibrium intermediate and transient intermediate of src SH3 and its mutant (2007)
  • Author(s): Jin Xianju, Li Jinsong, 松村 義隆, 新庄 正路, 笠井 恒希, 田代(下高原) 櫻子, 木原 裕
  • Organizer: 第45回日本生物物理学会年会
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-21
• [Presentation] パーキンソン病原因タンパク質alpha-synucleinのアミロイド線維形成の解析 (2007)
  • Author(s): 田代 充・田代 桜子・上吉原 智晃・小島 正樹・上田 健冶
  • Organizer: 第68回分析化学討論会
  • Place of Presentation: 宇都宮大学峰キャンパス
  • Year and Date: 2007-05-19
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Analysis of amyloid formation mechanism using alpha-synuclein from Parkinson's disease (2007)
  • Author(s): Tashiro, M., Shimotakahara, Tashiro, S., Kamiyoshihara, T., Kojima, M., Ueda, K
  • Organizer: 68th Analytical Chemistry Forum
  • Place of Presentation: University of Utsunomiya, Japan
  • Year and Date: 2007-05-19
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] パーキンソン病原因タンパク質a-synucleinのアミロイド線維形成の解析 (2007)
  • Author(s): 田代 櫻子, 上吉原 智晃, 小島 正樹, 上田 健治, 田代 充
  • Organizer: 日本薬学会第129年会
  • Place of Presentation: 富山
  • Year and Date: 2007-03-29
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Study of amyloid formation mechanism using alpha-synuclein from Parkinson's disease (2007)
  • Author(s): Shimotakahara, Tashiro, S., Kamiyoshihara, T., Kojima, M., Ueda, K., Tashiro, M.
  • Organizer: 129th Annual Pharmacy Society Meeting
  • Place of Presentation: Toyama, Japan
  • Year and Date: 2007-03-29
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] パーキンソン病関連タンパク質a-synucleinにおけるアミロイド形成機構の解明 (2005)
  • Author(s): 上吉原 智晃, 小島 正樹, 宮本 怜, 上田 健治, 田代 充, 神藤 季三郎, 下高原 桜子
  • Organizer: 第28回日本分子生物学会年会
  • Place of Presentation: 福岡
  • Year and Date: 2005-12-07
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Analysis of amyloid formation mechanism using alpha-synuclein from Parkinson's disease (2005)
  • Author(s): Kamiyoshihara, T., Kojima, M., Miyamoto, R., Ueda, K., Tashiro, M., Shindo, H., Shimotkahara, S
  • Organizer: 28th Annual Molecular Biology Society Meeting
  • Place of Presentation: Fukuoka, Japan
  • Year and Date: 2005-12-07
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 薬学生のための分析化学第3版 (2008)
  • Author(s): 岩木 和夫, 楠 文代, 渋澤 庸一, 田代 桜子, 田辺 顕子, 大和 進
  • Total Pages: 327
  • Publisher: 廣川書店
  • Description: 「研究成果報告書概要(和文)」より