Structure-inhibition of amyloid oligomers and fibrils

淀粉样蛋白寡聚体和原纤维的结构抑制

• 批准号: 7866473
• 负责人: STEVEN Owen SMITH
• 金额: $ 28.1万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7866473
• 关键词: AP40; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Fibrils; Amyloid beta-Protein; Architecture; Atomic Force Microscopy; Biological Assay; C-terminal; Cells; Data; Deposition; Electron Microscopy; Elements; Face; Fluorescence; Gene Mutation; Glycine; Image; In Vitro; Inherited; Methods; Modeling; Molecular Sieve Chromatography; Molecular Structure; NMR Spectroscopy; Neurodegenerative Disorders; Neurons; Peptides; Phenylalanine; Protein Isoforms; Proteins; Resolution; Sampling; Side; Solutions; Structural Models; Structure; Surface; Tail; Testing; Thioflavin T; Touch sensation; Toxic effect; abeta oligomer; amyloid fibril formation; base; design; dimer; inhibitor/antagonist; instrument; monomer; neuron loss; novel; novel strategies; prevent; research study; solid state nuclear magnetic resonance; three dimensional structure

DESCRIPTION (provided by applicant): Amyloid deposits found in neurodegenerative diseases result from misfolding of cellular proteins. The challenge for developing specific inhibitors that block oligomer or fibril formation is that there are no high- resolution molecular structures that can guide the design. The proposal has three specific aims. The first aim is to use high-resolution solid-state NMR in combination with atomic force microscopy (AFM) to refine the structures that are emerging of Abeta oligomers and fibrils. The second aim is to design inhibitors based on the structural data from Aim 1 and to test their ability in vitro to block oligomer or fibril formation. The third aim is to assay the ability of these inhibitors to block the toxicity of Abeta oligomers and fibrils on neuronal cells. A new approach has been developed for obtaining high-resolution AFM images of Abeta soluble oligomers and fibrils in solution. The method takes advantage of a novel AFM controller that provides resolution in 'single touch' AFM experiments that surpasses the resolution currently available using commercial instruments. High resolution AFM of solution samples will allow us to follow the formation of Abeta oligomers, protofibrils and fibrils, and determine how designed inhibitors prevent fibrillization. We have developed structural models for the Abeta42 monomer, dimer, protofibril and fibril based on preliminary results from high resolution AFM and solid-state NMR. The structures show that when the b-strands have a parallel orientation and the amino acids are in-register with one another, the surface of the b-sheet has pronounced ridges and grooves. This architecture provides the key elements for the rational design of inhibitors to prevent fibril formation. Our template inhibitor peptide based on a rational design approach has the sequence GxFxGxF, where the bulky phenylalanine side chains of the inhibitor are predicted to pack against the glycines in the GxxxG motif of the amyloidogenic peptide. We will test the ability of the designed peptides to disrupt the formation of oligomers and fibrils by thioflavin T fluorescence, size exclusion chromatography, electron microscopy, AFM and solid-state NMR. We will also test the ability of our designed inhibitors to protect neurons from cell death induced by amyloid fibrils. We will focus on the Abeta42 peptide because of its higher ability to form aggregates than the shorter isoforms. Moreover, most gene mutations that are associated with the inherited forms of Alzheimer's disease result in an increase in the ratio of Abeta42 over Abeta40.

描述（由申请人提供）：神经退行性疾病中发现的淀粉样蛋白沉积物是由细胞蛋白折叠折叠导致的。开发阻断低聚物或原纤维形成的特定抑制剂的挑战是，没有可以指导设计的高分辨率分子结构。该提案具有三个具体目标。第一个目的是将高分辨率固态NMR与原子力显微镜（AFM）结合使用，以完善正在出现Abeta低聚物和原纤维的结构。第二个目的是根据AIM 1的结构数据设计抑制剂，并在体外测试其能力阻断低聚物或原纤维形成的能力。第三个目的是测定这些抑制剂阻断Abeta低聚物和原纤维对神经元细胞的毒性的能力。 已经开发了一种新的方法来获得溶液中Abeta可溶性低聚物和原纤维的高分辨率AFM图像。该方法利用了一个新型的AFM控制器，该机构在“单触摸” AFM实验中提供了分辨率，该实验超过了当前使用商业仪器可用的分辨率。溶液样品的高分辨率AFM将使我们能够遵循Abeta低聚物，原纤维和原纤维的形成，并确定设计抑制剂如何防止纤维化。 我们根据高分辨率AFM和固态NMR的初步结果开发了Abeta42单体，二聚体，原纤维和原纤维的结构模型。结构表明，当B链具有平行方向并且氨基酸相互注册时，B表面的表面显着脊和凹槽。该体系结构为抑制剂的合理设计提供了预防原纤维形成的关键要素。我们的模板抑制剂肽基于有理设计方法具有GXFXGXF序列，其中预计抑制剂的庞大苯丙氨酸侧链被预测会在淀粉样蛋白生成肽的GXXXG基序中包裹甘氨酸。 我们将测试设计的肽通过硫非类T荧光，尺寸排除色谱，电子显微镜，AFM和固态NMR破坏寡聚和原纤维形成的能力。我们还将测试我们设计的抑制剂保护神经元免受淀粉样蛋白纤维诱导的细胞死亡的能力。我们将重点关注Abeta42肽，因为它比较短的同工型具有更高的形成聚集体能力。此外，大多数与阿尔茨海默氏病遗传形式相关的基因突变导致Abeta42比Abeta40的比率增加。