Structural Studies of Alzheimer's beta-Amyloid Fibrils

阿尔茨海默病 β-淀粉样原纤维的结构研究

• 批准号: 7593509
• 负责人: ROBERT TYCKO
• 金额: $ 33.82万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593509
• 关键词: Agitation; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Area; Biochemistry; Collaborations; Condition; Data; Electron Microscope; Fiber; Fluorescence; Genetic Polymorphism; Glutamine; Goals; Growth; Image; Label; Lead; Length; Localized; Manuscripts; Measurement; Modeling; Molecular; Molecular Conformation; Molecular Structure; Morphology; N-terminal; National Institute of Diabetes and Digestive and Kidney Diseases; Pathway interactions; Peptide Conformation; Preparation; Prions; Protein Structure Initiative; Publishing; Scanning Transmission Electron Microscopy Procedures; Science; Seeds; Solutions; Structural Models; Structure; Structure of molecular layer of cerebellar cortex; System; Variant; Work; Yeasts; amyloid formation; amyloid peptide; base; beta pleated sheet; molecular modeling; neurotoxicity; neutrophil; new growth; physical chemical interaction; research study; solid state

Progress in FY2007 has been in the following areas: 1. STRUCTURAL MODEL FOR THREE-FOLD SYMMETRIC BETA-AMYLOID FIBRILS: In earlier work (Petkova et al., Science 2005), we discovered that the 40-residue beta-amyloid peptide can form more than one fibril structure, that the predominant fibril structure can be determined by subtle variations in growth conditions, that fibrils with distinct morphologies in electron microscope images have distinct underlying molecular structures, and that morphology and molecular structure are self-propagating when pre-existing fibril fragments are used as seeds for the growth of new fibrils. In earlier work (Petkova et al., Biochemistry 2006), we have also developed a full structural model for beta-amyloid fibrils that form under gentle agitation of a beta-amyloid solution and that have mass-per-length (MPL) values, determined by scanning transmission electron microscopy (collaboration with R.D. Leapman, DBEPS, NIBIB), corresponding to two molecular layers in a cross-beta structural motif. We found that fibrils grown under quiescent solution conditions have MPL values that correspond to THREE molecular layers, but our earlier solid state NMR data indicated that two or more quiescent fibril structures might coexist. Now, in FY2007, we have succeeded in preparing highly structurally homogeneous beta-amyloid fibrils with MPL values corresponding to three molecular layers, and we have succeeded in obtaining sufficient structural constraints from solid state NMR to develop a full molecular model. Whereas our previously published model for agitated fibrils had two-fold symmetry about the long fibril axis, the new model (which applies to a different fibril type) has three-fold symmetry. The beta-amyloid peptide conformations in two-fold and three-fold symmetric structures are similar, with essentially the same beta-strand segments and the same in-register parallel beta-sheet tertiary structure, but the conformations of non-beta-strand segments are different, and the contacts between beta-sheets are also different. A manuscript describing the new beta-amyloid fibril structure is currently in preparation. Additional measurements on three-fold symmetric fibrils are in progress, including cryo-EM studies in collaboration with U. Baxa and A.C. Steven of NIAMS. 2. In collaboration with R.B. Wickner's group in NIDDK, we have carried out related studies of amyloid fibrils formed by the yeast prion proteins Ure2p and Sup35p. These are the first solid state NMR studies of full-length prion domains of yeast prions. In experiments on Sup35NM (the N-terminal and middle domains of Sup35p), we showed that Sup35NM fibrils are constructed from in-register parallel beta-sheets and do not have a beta-helical structure, which had been suggested by earlier x-ray fiber diffraction and fluorescence labeling experiments in other groups. Interestingly, our solid state NMR data indicate that the beta-sheets in Sup35NM are not localized exclusively in the N-terminal, Asn- and Gln-rich domain (which was expected to form the amyloid fibril core), but are also found in parts of the M domain. This work has been published in PNAS, vol. 103, p. 19754, year 2006. Beta-sheet formation in the M domain may be the distinguishing structural feature among PSI prion variants or strains, which are induced by intracellular Sup35p amyloid formation. This possibility is supported by recent H/D exchange data from the Weissman group at UCSF, which indicate different degrees of H/D exchange protection in the M domain for different Sup35NM amyloid variants. We have also carried out solid state NMR studies of the prion domain (residues 1-89) of Ure2p. Ure2p(1-89) fibrils also contain in-register parallel beta-sheets. This work is described in a manuscript that is currently under review for Biochemistry.

Progress in FY2007 has been in the following areas: 1.三重对称β-淀粉样蛋白原纤维的结构模型：在早期工作中（Petkova等人，Science 2005），我们发现40个残基的β-淀粉样肽可以形成多个原纤维结构，主要的原纤维结构可以通过生长条件的细微变化来确定，电子显微镜图像中具有不同形态的原纤维具有不同的结构。底层的 分子结构，并且当预先存在的原纤维片段用作新原纤维生长的种子时，形态和分子结构是自我繁殖的。 在早期工作中（Petkova 等人，Biochemistry 2006），我们还开发了 β-淀粉样蛋白原纤维的完整结构模型，该模型在 β-淀粉样蛋白溶液的温和搅拌下形成，并具有通过扫描透射电子显微镜（与 R.D. Leapman、DBEPS、NIBIB 合作）确定的每长度质量 (MPL) 值，对应于交叉 β 淀粉样蛋白中的两个分子层 结构主题。 我们发现在静态溶液条件下生长的原纤维具有对应于三个分子层的 MPL 值，但我们早期的固态 NMR 数据表明两个或多个静态原纤维结构可能共存。 现在，在2007财年，我们已经成功制备了结构高度均质的β-淀粉样蛋白原纤维，其MPL值对应于三个分子层，并且我们已经成功地从固态NMR获得了足够的结构约束来开发完整的分子模型。 虽然我们之前发布的搅动原纤维模型关于长原纤维轴具有二重对称性，但新模型（适用于不同的原纤维类型）具有三重对称性。 二重和三重对称结构的β淀粉样肽构象相似，具有基本相同的β链片段和相同的对齐平行β折叠三级结构，但非β链片段的构象不同，β折叠之间的接触也不同。 A manuscript describing the new beta-amyloid fibril structure is currently in preparation. 对三重对称原纤维的其他测量正在进行中，包括与 NIAMS 的 U. Baxa 和 A.C. Steven 合作进行的冷冻电镜研究。 2. 我们与NIDDK的R.B. Wickner课题组合作，对酵母朊病毒蛋白Ure2p和Sup35p形成的淀粉样原纤维进行了相关研究。 These are the first solid state NMR studies of full-length prion domains of yeast prions. 在Sup35NM（Sup35p的N端和中间结构域）的实验中，我们表明Sup35NM原纤维是由对齐的平行β-折叠构建的，并且不具有β-螺旋结构，这一点已被其他组的早期X射线纤维衍射和荧光标记实验所暗示。 有趣的是，我们的固态 NMR 数据表明 Sup35NM 中的 β-折叠不仅仅位于 N 末端、富含 Asn 和 Gln 的结构域（预计会形成淀粉样原纤维核心），而且也存在于 M 结构域的部分区域。 这项工作已发表在《PNAS》，第 1 卷。 103，p。 19754，2006 年。M 结构域中的 β 片层形成可能是 PSI 朊病毒变体或菌株的显着结构特征，其由细胞内 Sup35p 淀粉样蛋白形成诱导。 UCSF Weissman 小组最近的 H/D 交换数据支持了这种可能性，该数据表明不同 Sup35NM 淀粉样蛋白变体的 M 结构域中存在不同程度的 H/D 交换保护。 We have also carried out solid state NMR studies of the prion domain (residues 1-89) of Ure2p. Ure2p(1-89) fibrils also contain in-register parallel beta-sheets. This work is described in a manuscript that is currently under review for Biochemistry.