Structural Biology of Amyloid Beta-Protein

描述(由申请人提供)：我们假设淀粉样蛋白(A)组装成神经毒性寡聚体和聚合物是阿尔茨海默病(AD)的一个原始神经病理过程。如果是这样的话，抑制或解离现有的组装可能是有效的治疗方法。为了验证我们的假设，必须详细阐明A？的结构生物学。低聚物的构象是什么？通过什么机制？这样形成的低聚物的结构是什么？每种齐聚物的相对毒性是什么？许多人，包括我们自己，都在努力将结构与生物活性的衡量标准联系起来。最近的研究表明，二聚体或三聚体是重要的神经毒素，但六聚体、非聚体、十二聚体和较大的寡聚体也被证明是有效的神经毒素。这项建议的长期目标是从简单的四级结构测定转向阐明单体的二级和三级结构动力学以及单体齐聚机理的确定。这意味着最终理解控制动力学的原子间相互作用，并在这样做的过程中，以原子分辨率识别治疗靶点。这种“以知识为基础”的方法有别于高通量筛查策略，但与之互补。这两种方法都应该被执行，以最大限度地增加识别有效的、改变疾病的治疗剂的机会。我们建议：(1)阐明A？单体折叠和自组装的物理生物化学；(2)建立由此形成的A？集合体的结构-神经毒性关系。为此，我们将通过化学方法合成特定氨基酸和化学键发生变化的多肽，然后研究这些多肽的构象动力学和组装。这些改变的位置和改变本身都经过了仔细的选择，以揭示A分子的关键结构特征，这些结构特征控制着A？分子组装成损伤或杀死神经元的结构。我们将识别、分离和结构表征特定类型的组件，然后通过处理培养中的原代神经元来定量确定每个组件的毒性活性。在第一个目标中获得的对大会结构的了解的深度将是前所未有的。因此，通过这种“结构-活性关联”过程获得的知识有望提供对哪些组件以及这些组件上的哪些结构(在原子分辨率下)应作为治疗靶点的最准确评估。除了有助于提高对阿尔茨海默病及其治疗的理解外，拟议项目的结果还应与其他与蛋白质组装异常有关的神经退行性疾病的研究相关。这些疾病包括帕金森氏症、亨廷顿氏症、肌萎缩侧索硬化症、家族性淀粉样多发性神经病和红斑狼疮。

DESCRIPTION (provided by applicant): We hypothesize that amyloid ¿-protein (A¿) assembly into neurotoxic oligomers and polymers is a seminal neuropathogenetic process in Alzheimer's disease (AD). If so, assembly inhibition or dissociation of existing assemblies could be effective therapeutic approaches. To test our hypothesis, the structural biology of A¿ must be elucidated in detail. What conformers form oligomers? By what mechanism? What are the structures of the oligomers thus formed? What is the relative toxicity of each oligomer species? Many, including ourselves, have striven to correlate structure with measures of biological activity. Recent work has suggested that dimeric or trimeric assemblies are important neurotoxins, but hexameric, nonameric, dodecameric, and larger oligomers also have been shown to be potent neurotoxins. The long-term goal of this proposal is to move past simple quaternary structure determination to elucidation of A¿ monomer secondary and tertiary structure dynamics and the determination of mechanisms of monomer oligomerization. This means eventually understanding the interatomic interactions that control the dynamics, and in doing so, identifying therapeutic targets at atomic resolution. This "knowledge-based" approach is distinct from, but complementary to, high-throughput screening strategies. Both approaches should be executed to maximize the chances for identifying efficacious, disease-modifying therapeutic agents. We propose here to: (1) elucidate the physical biochemistry of A¿ monomer folding and self-assembly; and (2) establish structure-neurotoxicity relationships of the A¿ assemblies thus formed. To do so, we will chemically synthesize A¿ peptides in which specific amino acids and chemical bonds are altered and then study the conformational dynamics and assembly of these peptides. The positions of these alterations, and the alterations themselves, have been chosen carefully so as to reveal the key structural features of the A¿ molecule that control its assembly into structures that damage or kill neurons. We will identify, isolate, and structurally characterize specific types of assemblies and then determine quantitatively the toxic activity of each assembly by treating primary neurons in culture. The depth of understanding of the structures of the assemblies obtained in the first aim will be unprecedented. Thus the knowledge gained through this "structure-activity correlation" process is expected to provide the most accurate assessment of which assemblies, and which structures (at atomic resolution) on these assemblies, should be targeted therapeutically. In addition to its contributions to an improved understanding of AD and its treatment, results of the proposed project should have relevance for studies of other neurodegenerative diseases linked to aberrant protein assembly. These include Parkinson's, Huntington's, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and the prionoses.