Structures and Toxicity of Amyloid Protein Assemblies in Alzheimer's

描述(申请人提供)：这项研究的长期目标是通过研究阿尔茨海默病(AD)中常见的三种类型的阿尔茨海默氏症-淀粉样蛋白(A？)的剧毒蛋白质组合的分子细节，确定在阿尔茨海默病(AD)中观察到的神经细胞死亡的根源。大脑中老年斑的形成是阿尔茨海默病(AD)的标志；斑块的主要成分是由A？组成的纤维集合体。由于A？通过自组装成更大的聚集形式(即单体A？是无毒的)而表现出毒性，长期以来人们一直怀疑A？的错误折叠导致A？通过自组装形成纤维和结构变化，从而触发AD的毒性和神经功能障碍的发生。事实上，聚合体A的毒性很大程度上受组装的A的形态、序列的细微差异以及特定配体的存在(如铜(II))的影响。在这项研究中，我们将用固态核磁共振(SS核磁共振)检测A？的三种不同形式的有毒淀粉样聚集体的原子级结构，这已被用作包括A？的淀粉样纤维结构分析的主要工具。在目标1中，我们将检验一个流行的假设，即A？的毒性与铜(II)与A？聚集体的结合有关。人们普遍认为，与铜(II)结合的淀粉样蛋白聚集体可能催化产生过氧化氢的反应，这对神经细胞是有毒的。然而，由于纤维的内在异质性，传统的结构生物学方法如X射线结晶学和溶液核磁共振等不能有效地分析金属结合结构。通过SS核磁共振，我们将研究铜(II)与A的结合位置和方式，以及由于结合而产生的任何结构变化。在目标2中，我们针对A？(1-42)的淀粉样纤维和可扩散的亚纤维聚集体的结构，尽管它们具有致病作用，但由于样品制备的极端困难，这些结构的特征一直很差。这些淀粉样蛋白纤维和A？(1-42)中间体的分子结构引起了广泛的关注，因为这些结构为设计新的治疗方法和早期发现AD提供了洞察力。我们将通过使用一种新的灵敏度增强方法来克服样品制备问题，这种方法可以最大限度地减少SS核磁共振所需的样本量。这些研究将通过SS核磁共振揭示高度神经毒性的淀粉样原纤维和A？(1-42)中间体的第一个特定部位的结构细节。在目标3中，我们表征了E22G A？(1-40)的错误折叠结构和动力学，它是一种独特的致病突变体，可以促进亚纤维聚集体的形成。我们的研究旨在首次揭示E22G A？(1-40)纤维和亚纤维聚集体的特定部位构象。相关淀粉样聚集体的神经毒性将在小鼠PC12细胞上进行检测。

DESCRIPTION (provided by applicant): The long-term objective of this research is to identify the origin of neural cell deaths observed in Alzheimer's disease (AD) through investigating molecular details of three types of highly toxic protein assemblies of Alzheimer's ¿-amyloid (A¿), which are commonly observed in AD. The formation of senile plaques in a brain is a hall mark of Alzheimer's disease (AD); the primary component of the plaque is fibrillar assemblies comprised of A¿. Because A¿ exhibits toxicity through self-assembly into larger aggregated forms (i.e. monomeric A¿ is non-toxic), it has been long suspected that misfolding of A¿ resulting in the fibril formation and structural changes of A¿ via the self-assembly trigger the onset of the toxicity and the neural dysfunctions in AD. Indeed, the toxicity of the aggregated A¿ is greatly modulated by morphologies of the assembled A¿, subtle difference in the sequence, and the presence of particular ligands such as Cu(II). In this research, we will examine atomic-level structures of three distinctive forms of toxic amyloid aggregates for A¿ by solid-state NMR (SSNMR), which has been used as a primary tool in structural analysis for amyloid fibrils, including those for A¿. In Aim 1, we will examine a popular hypothesis that the toxicity of A¿ is associated with Cu(II) binding to A¿ aggregates. It is widely believed that Cu(II)-bound amyloid aggregates may catalyze reactions producing H2O2, which is toxic to neural cells. However, because of the intrinsic heterogeneity of the fibrils, traditional methods in structural biology such as X-ray crystallography and solution NMR are not effective for analysis of the metal-binding structures. With SSNMR, we will examine the location and the mode of Cu(II) binding to A¿ as well as any structural changes due to binding. In Aim 2, we target the structure of amyloid fibrils and diffusible subfibrillar aggregates for A¿(1-42), which have been poorly characterized, despite their pathogenic importance, because of the extreme difficulties in sample preparation. Molecular structures of these amyloid fibrils and intermediates for A¿(1-42) have attracted broad attention since the structures offer insights into designs of new therapies and early detection for AD. We will overcome the sample preparation problems by using a novel sensitivity enhancement method, which minimizes sample amount required for SSNMR. The studies will reveal the first site-specific structural details of highly neurotoxic amyloid fibril and intermediate for A¿(1-42) by SSNMR. In Aim 3, we characterize structure and kinetics in misfolding for E22G A¿(1-40), which is a unique pathogenic mutant that promotes formation of subfibrillar aggregates. Our study aims to reveal site-specific conformations for E22G A¿(1-40) fibrils and subfibrillar aggregates for the first time. The neural toxicity of the relevant amyloid aggregates will be examined on mouse PC12 cells.