Structure-inhibition of amyloid oligomers and fibrils

描述(申请人提供)：在神经退行性疾病中发现的淀粉样沉积是细胞蛋白质错误折叠的结果。开发阻断低聚物或纤维形成的特定抑制剂的挑战是，没有高分辨率的分子结构可以指导设计。该提案有三个具体目标。第一个目标是使用高分辨率固态核磁共振结合原子力显微镜(AFM)来精炼正在出现的Aβ齐聚物和纤维的结构。第二个目标是根据目标1的结构数据设计抑制剂，并在体外测试它们阻断低聚物或纤维形成的能力。第三个目的是测试这些抑制剂阻断Abeta寡聚体和纤维对神经细胞的毒性的能力。 发展了一种获得溶液中Abeta可溶低聚物和纤维的高分辨率AFM图像的新方法。该方法利用了一种新型的原子力显微镜控制器，该控制器在“单触式”原子力显微镜实验中提供的分辨率超过了目前使用商业仪器提供的分辨率。溶液样品的高分辨率AFM将使我们能够跟踪Abeta齐聚物、原纤维和纤维的形成，并确定设计的抑制剂如何防止纤化。 基于高分辨率AFM和固体核磁共振的初步结果，我们开发了Abeta42单体、二聚体、原纤维和纤维的结构模型。结构表明，当b-链具有平行取向并且氨基酸相互对准时，b-折叠的表面具有明显的脊状和凹槽。这一体系结构为合理设计抑制剂以防止纤维形成提供了关键要素。我们的模板抑制物肽基于合理的设计方法，具有序列GxFxGxF，其中抑制物的笨重的苯丙氨酸侧链被预测为与淀粉样肽的GxxxG基序中的甘氨酸相反。 我们将通过硫代黄素T荧光、尺寸排除层析、电子显微镜、原子力显微镜和固体核磁共振来测试设计的多肽破坏低聚物和纤维形成的能力。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，因为它比较短的异构体形成聚集体的能力更强。此外，与阿尔茨海默病的遗传形式相关的大多数基因突变都会导致Abeta42比Abeta40的比例增加。

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.