Structures and Toxicity of Amyloid Protein Assemblies in Alzheimer's

描述（由申请人提供）：本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高毒性蛋白质组装体的分子细节，确定阿尔茨海默病（AD）中观察到的神经细胞死亡的起源。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？组成的纤维状组装体。因为A通过自组装成更大的聚集形式表现出毒性（即单体A <$是无毒的），长期以来一直怀疑A <$的错误折叠导致原纤维形成和A <$通过自组装的结构变化触发了AD中毒性和神经功能障碍的发作。事实上，聚集的A <$的毒性受到组装的A <$的形态、序列的细微差异以及特定配体如Cu（II）的存在的极大调节。 在这项研究中，我们将通过固态核磁共振（SSNMR）研究三种不同形式的有毒淀粉样蛋白聚集体的原子级结构，固态核磁共振已被用作淀粉样蛋白原纤维结构分析的主要工具，包括A？。在目标1中，我们将研究一个流行的假设，即A <$的毒性与Cu（II）结合A <$聚集体有关。人们普遍认为，Cu（II）结合的淀粉样蛋白聚集体可以催化产生H2 O2的反应，H2 O2对神经细胞有毒。然而，由于原纤维的内在异质性，结构生物学中的传统方法，如X射线晶体学和溶液NMR，不能有效地分析金属结合结构。与SSNMR，我们将检查的位置和模式的Cu（II）结合到A的，以及任何结构的变化，由于结合。在目标2中，我们针对A？（1-42）的淀粉样蛋白原纤维和可扩散的亚原纤维聚集体的结构，尽管它们具有致病重要性，但由于样品制备的极端困难，它们的特征很差。这些淀粉样蛋白原纤维和A？（1-42）中间体的分子结构引起了广泛的关注，因为这些结构为AD的新疗法设计和早期检测提供了见解。我们将克服样品制备问题，通过使用一种新的灵敏度增强方法，最大限度地减少SSNMR所需的样品量。这些研究将通过SSNMR揭示高神经毒性淀粉样蛋白原纤维和A？（1-42）中间体的第一个位点特异性结构细节。在目标3中，我们表征了E22 G A？（1-40）错误折叠的结构和动力学，E22 G A？（1-40）是一种独特的致病突变体，可促进亚纤维聚集体的形成。我们的研究旨在首次揭示E22 GA <$（1-40）原纤维和亚原纤维聚集体的位点特异性构象。将在小鼠PC 12细胞上检查相关淀粉样蛋白聚集体的神经毒性。 公共卫生相关性：该项目提供了神经毒性蛋白质的详细分子结构，这些蛋白质可能在阿尔茨海默病的发展中发挥核心作用。这项研究将为阿尔茨海默氏症药物的合理设计提供见解。

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. PUBLIC HEALTH RELEVANCE: This project provides detailed molecular structure for neurotoxic proteins, which are likely to play central roles in development of Alzheimer's disease. The research will provide insights into rational designs of drugs for Alzheimer's.