Elucidatiop of Tangle Formation Mechanism Based on 3D-structural Analysis of Tau Protein

基于 Tau 蛋白 3D 结构分析阐明缠结形成机制

• 批准号: 13680752
• 负责人: ISHIDA Toshimasa
• 金额: $ 2.18万
• 依托单位: Osaka University of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680752/
• 关键词: tau protein; formation mechanism of tangle; microtubule-binding domain; 3D-structure; X-ray analysis; NMR; Spectroscopic analysis; molecular modeling; タングル形式

1)X-ray crystal structure analysis of microtubule-binding domainTo elucidate 3D structure of tau protein, we attempted various crystallizations of tau and its fragments. Consequently, we succeeded in the crystallization of tau microtubule-binding domain (MBD) as GST fusion protein, which were prepared as recombinant by gene expression from E.coli. The crystal data are : tetragonal, space group P4_32_12, a=b=92.24Å, c=57.68Å. X-ray diffraction data up to 2Å resolution were collected by synchrotron of Spring 8. The crystal structure was solved by the molecular replacement method using the 3D structure of GST. After several structure refinement, the present R factor is 0.258. Although the MBDs formed dimeric structure, they showed large conformational flexibility and their atomic positions were not determined. This is in contrast with the rigid 3D structure of GST. Since the MBD constructs the core structure of tau PHF, the present result indicates the close relationship between the confo … More rmational flexibility of MBD and PHE formation. We are now in progress in analyzing the crystal structure more accurately.2)Analyses of solution structure of MBDMBD is composed of three-or four-repeated structure, where each repeat peptide (named R1 -R4) consists of 31 or 32 amino acid residues. To elucidate the conformational flexibility of each repeat peptide, the solution structures of R1 -R4 in TEE solution were analyzed by the combination of NMR and model building. Consequently, the the structures of R1, R2 and R4 peptides showed α-helical structure of amphipathic distribution of respective amino acid residues. In contrast, R3 took both of extended and α-helical structures of amphipathic behavior. This result suggest the molecular association of MBD through alternative hydrophilic and hydrophobic interactions.3)Investigation of MBD interactions and model of repeat-structure-dependent PHF formationThe association of neighboring MBDs were investigated by ThS fluorescence, light scattering and surface plasmon resonance analyses, and electromicroscopy. On the basis of these results, we proposed the first model of repeat structure-controlled step-by-step filament formation of 4RMBD. This model would be helpful upon considering rational approaches to prevent tau deposition in AD Less

1）微管结合区的X射线晶体结构分析为了阐明tau蛋白的三维结构，我们尝试了tau蛋白及其片段的各种结晶。因此，我们成功地结晶tau微管结合结构域（MBD）作为GST融合蛋白，这是通过基因表达从大肠杆菌中制备的重组体。晶体数据为：四氢呋喃，空间群P4_32_12，a=B=92.24，c=57.68。X-射线衍射数据高达2 μ m的分辨率，收集同步加速器的春天8。利用GST的三维结构，通过分子置换法求解了晶体结构。经过多次结构优化，目前的R因子为0.258。虽然MBDs形成二聚体结构，但它们表现出较大的构象灵活性，并且它们的原子位置无法确定。这与GST的刚性3D结构形成对比。由于MBD构建了tau PHF的核心结构，因此本研究结果表明，tau PHF的构象与其蛋白质结构之间存在密切关系。 ...更多信息 MBD和PHE形成的相对灵活性。2）MBDMBD的溶液结构分析表明，MBDMBD由三个或四个重复序列组成，每个重复序列（R1 -R4）由31或32个氨基酸残基组成。为了阐明每个重复肽的构象灵活性，通过核磁共振和模型建立相结合的方法分析了R1 -R4在TEE溶液中的溶液结构。因此，R1、R2和R4肽段的结构均为α-螺旋结构，氨基酸残基呈两亲性分布。而R3则同时具有两亲性的α-螺旋和伸展螺旋结构。3）MBD相互作用的研究和重复结构依赖的PHF形成模型通过ThS荧光、光散射和表面等离子体共振分析以及电子显微镜研究了相邻MBD之间的缔合作用。在此基础上，我们提出了第一个由重复结构控制的4 RMBD逐步成丝模型。该模型将有助于考虑合理的方法来防止AD Less中的tau沉积

项目成果

T.-M.Yao, K.Tomoo, T.Ishida, M.Sumida, M.sasaki, T.Taniguchi: "Aggregation analysis of the microtubule binding domain in tau protein by spectroscopic method"Peptide Science. 2002. 249-252 (2003)

T.-M.Yao、K.Tomoo、T.Ishida、M.Sumida、M.sasaki、T.Taniguchi：“通过光谱法对 tau 蛋白中微管结合域进行聚集分析”肽科学。

K.Minoura, K.Tomoo, T.Ishida, H.Hasegawa, M.Sasaki, T.Taniguchi: "Solvent-dependent conformation of the third repeat fragment in the microtubule-binding domain of tau protein, analyzed by 1H-NMR spectroscopy and molecular modeling calculation"Bull.Chem.So

K.Minoura、K.Tomoo、T.Ishida、H.Hasekawa、M.Sasaki、T.Taniguchi：“tau 蛋白微管结合域中第三个重复片段的溶剂依赖性构象，通过 1H-NMR 光谱分析

S.Hiraoka, T.-M.Yao, K.Minoura, K.Tomoo, M.Sumida, T.Taniguchi, T.Ishida: "Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein"Biochem.Biophys.Res.Commun.. 315. 659-663 (2004)

S.Hiraoka、T.-M.Yao、K.Minoura、K.Tomoo、M.Sumida、T.Taniguchi、T.Ishida：“构象过渡态负责 tau 蛋白微管结合域的组装”Biochem。

K.Minoura, K.Tomoo, Y.In, T.Ishida, H.Hasegawa, M.Sasaki, T.Taniguchi: "Amphipathic helical behavior of the third repeat fragment in the tau microtuble-binding domain, studied by 1H-NMR spectroscopy"Biochem.Biophys.Res.Commun.. 294. 210-214 (2002)

K.Minoura、K.Tomoo、Y.In、T.Ishida、H.Hasekawa、M.Sasaki、T.Taniguchi：“tau 微管结合域中第三个重复片段的两亲螺旋行为，通过 1H-NMR 研究

T.-M.Yao, K.Tomoo, Y.In, T.Ishida, M.Sumida, M.sasaki, T.Taniguchi: "Aggregation analysis of microtubule binding domain in tau protein by spectroscopic method"Peptide Science. 2002. 249-252 (2003)

T.-M.Yao、K.Tomoo、Y.In、T.Ishida、M.Sumida、M.sasaki、T.Taniguchi：“通过光谱法对 tau 蛋白中微管结合域进行聚集分析”肽科学。