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）中的一个重要神经病理过程。如果是这样的话，组装抑制或解离现有的组件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。什么构象形成低聚物？通过什么机制？由此形成的低聚物的结构是什么？每种低聚物的相对毒性是多少？许多人，包括我们自己，一直在努力将结构与生物活动的测量相关联。最近的研究表明，二聚体或三聚体组装体是重要的神经毒素，但六聚体，九聚体，十二聚体和更大的寡聚体也已被证明是有效的神经毒素。该提案的长期目标是通过简单的四级结构测定来阐明A？单体二级和三级结构动力学以及单体低聚反应机制的测定。这意味着最终了解控制动力学的原子间相互作用，并在此过程中，以原子分辨率确定治疗靶点。这种“基于知识”的方法与高通量筛选策略不同，但又是其补充。这两种方法都应该执行，以最大限度地提高识别有效的，改善疾病的治疗药物的机会。我们在此提议：（1）阐明A <$单体折叠和自组装的物理生物化学;（2）建立由此形成的A <$组装体的结构-神经毒性关系。为此，我们将化学合成A肽，其中特定的氨基酸和化学键被改变，然后研究这些肽的构象动力学和组装。这些改变的位置，以及改变本身，都是经过仔细选择的，以揭示A？分子的关键结构特征，这些特征控制着A？分子组装成损伤或杀死神经元的结构。我们将识别，隔离，并在结构上表征特定类型的组件，然后通过处理培养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组件结构的理解的深度将是前所未有的。因此，通过这种“结构-活性相关性”过程获得的知识预计将提供对哪些组装体以及这些组装体上的哪些结构（原子分辨率）应该在治疗上靶向的最准确的评估。除了有助于提高对AD及其治疗的理解外，拟议项目的结果还应与与异常蛋白质组装相关的其他神经退行性疾病的研究相关。这些疾病包括帕金森氏病、亨廷顿病、肌萎缩侧索硬化症、家族性淀粉样多发性神经病和朊病毒病。 公共卫生相关性：该项目将促进我们对蛋白质，淀粉样蛋白，如何导致阿尔茨海默病的理解。这种理解可以直接转化为一类新药物的开发，这些药物有可能改变或治愈这种疾病。该项目还将与帕金森病、亨廷顿病、肌萎缩侧索硬化症、家族性淀粉样多发性神经病、朊病毒病和其他衰老神经退行性疾病有关。

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. PUBLIC HEALTH RELEVANCE: This project will advance our understanding of how a protein, the amyloid-protein, causes Alzheimers disease. This understanding can be translated directly into the development of a new class of drugs that have the potential to modify or cure the disease. The project also will be of relevance to Parkinsons, Huntingtons, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, the prionoses, and other neurodegenerative diseases of aging.