Structure-inhibition of amyloid oligomers and fibrils

描述（由申请方提供）：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质的错误折叠引起的。开发阻断低聚物或原纤维形成的特异性抑制剂的挑战在于没有可以指导设计的高分辨率分子结构。该提案有三个具体目标。第一个目标是使用高分辨率固态NMR结合原子力显微镜（AFM）来细化Abeta寡聚体和原纤维的结构。第二个目的是基于来自目的1的结构数据设计抑制剂，并测试它们在体外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寡聚体和原纤维对神经元细胞的毒性的能力。 一种新的方法已被开发用于获得高分辨率的AFM图像的Abeta可溶性低聚物和原纤维在溶液中。该方法利用了一种新型的AFM控制器，该控制器在“单触摸”AFM实验中提供的分辨率超过了目前使用商业仪器的分辨率。溶液样品的高分辨率AFM将使我们能够跟踪Abeta寡聚体，原纤维和原纤维的形成，并确定设计的抑制剂如何防止纤维化。 我们已经开发了结构模型的Abeta42单体，二聚体，原纤维和原纤维的基础上，从高分辨率AFM和固态NMR的初步结果。结构表明，当b链具有平行取向并且氨基酸彼此对齐时，b片层的表面具有明显的脊和槽。这种结构为合理设计抑制剂以防止原纤维形成提供了关键要素。我们基于合理设计方法的模板抑制剂肽具有序列GxFxGxF，其中预测抑制剂的大体积苯丙氨酸侧链与淀粉样蛋白生成肽的GxxxG基序中的甘氨酸包装。 我们将测试所设计的肽的能力，破坏形成的低聚物和纤维的硫磺素T荧光，尺寸排阻色谱法，电子显微镜，原子力显微镜和固态NMR。我们还将测试我们设计的抑制剂保护神经元免受淀粉样纤维诱导的细胞死亡的能力。我们将关注Abeta42肽，因为它比较短的同种型形成聚集体的能力更高。此外，大多数与阿尔茨海默病遗传形式相关的基因突变导致A β 42与A β 40的比率增加。

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.