Amyloid ion channels to design therapeutics for neurodegenerative diseases

描述（由申请人提供）：异常（或错误）折叠将蛋白质的3D构象从天然（可溶性形式）改变为非天然（不溶于）多态性淀粉样蛋白结构。蛋白质错误折叠与神经变性（阿尔茨海默氏症、亨廷顿氏症、帕金森氏症、家族性痴呆、朊病毒脑病）、系统性（II型糖尿病、轻链淀粉样变性相关的癌症）和其它（囊性纤维化）疾病有关。普遍的观点认为，蛋白质错误折叠诱导的淀粉样蛋白导致功能获得，并通过破坏细胞离子稳态引起病理生理细胞反应。理解蛋白质错误折叠和由此产生的诱导病理生理活性的3D构象一直是一个重要但具有挑战性的研究领域。淀粉样蛋白纤维形成及其预防的机制正在被广泛研究，尽管淀粉样蛋白纤维似乎并不直接引起神经退行性疾病;最近的研究表明，只有球状淀粉样蛋白足以引起病理生理反应。球状寡聚体介导的毒性的最直接机制将涉及它们的膜穿孔作为关键的初始事件。因此，我们普遍认为蛋白质错误折叠疾病是由小球状淀粉样蛋白形成离子通道破坏细胞离子稳态引起的。因此，调节其通道结构和活性的分子和其他干预措施可以用于有效的治疗。事实上，淀粉样蛋白生成肽在天然细胞以及人工膜中诱导离子电导。膜结合的淀粉样蛋白复合物的结构研究受到限制。我们的研究表明，与多种疾病相关的淀粉样肽形成多态性离子通道。这项持续的提议主要集中在阿尔茨海默病（AD）相关的淀粉样蛋白β（Ab）肽，形成有毒通道。我们打算定义3D结构多态性，并确定氨基酸（AA）表位的抗体肽，然后可以用作设计有效的治疗AD的目标。我们将继续我们的多维和互补的方法，AFM成像，离子电导记录，分子动力学（MD）模拟，细胞钙的摄取和变性，以获得淀粉样蛋白离子通道的全面了解。我们的具体目标是：1）对合成的以及组织衍生的淀粉样蛋白生成肽和在脂质膜中重构的具有位点特异性氨基酸（AA）取代的肽的3D结构进行成像; 2）成像由正常的以及具有位点特异性氨基酸（AA）取代的肽制成的通道响应于药理学试剂的开闭构象，3）使用集成离子电导AFM和药理学试剂以及位点特异性AA取代将开闭通道构象与离子电导相关联，以及4）检查细胞效应（例如，钙摄取和变性）。我们的研究将确定特定的淀粉样蛋白结构的基础阿尔茨海默氏症和其他退行性病理生理学，并将确定特定的结构基序（S）的淀粉样蛋白离子通道，然后可以用于设计药理学干预的治疗价值。

DESCRIPTION (provided by applicant): Abnormal (or mis-)folding alters protein's 3D conformation from native (soluble form) to non-native (insoluble from) polymorphic amyloid structures. Protein misfolding is linked to neurodegenerative (Alzheimer's, Huntington's, Parkinson's, familial dementia, prion encephalopathies), systemic (type II diabetes, light chain amyloidosis related cancer) and other (cystic fibrosis) diseases. Prevailing view suggests that protein misfolding-induced amyloids result into a gain-of-function and cause pathophysiologic cell response by destabilizing cell ionic homeostasis. Understanding protein misfolding and the resulting 3D conformations that induce pathophysiologic activity have been an important but challenging area of research. Mechanisms underlying amyloid fibril formation and its prevention are being studied extensively although amyloid fibers do not directly appear to cause neurodegenerative diseases; recent studies have shown that only globular amyloids are sufficient to cause pathophysiologic responses. The most direct mechanism of globular oligomer- mediated toxicity would involve their membrane poration as the key initial events. Our prevailing paradigm, therefore, is that protein misfolding diseases result from small globular amyloids forming ion channels to destabilize cell ionic homeostasis. Molecules and other interventions that modulate their channel structure and activity could thus be used for effective therapy. Indeed, amyloidogenic peptides induce ionic conductances in both native cell as well as artificial membranes. Structural study of membrane-bound amyloid complexes has been limited. Our studies have shown that amyloid peptides associated with several diseases form polymorphic ion channels. This continuing proposal primarily focuses on Alzheimer's disease (AD) linked amyloid beta (Ab) peptide that forms toxic channels. We intend to define the 3D structural polymorphism and identify amino acid (AA) epitopes in Ab peptide that can then be used as targets for designing effective therapeutics for AD. We will continue our multidimensional and complementary approaches of AFM imaging, ion conductance recording, molecular dynamics (MD) simulation, and cell Calcium uptake and degeneration to obtain a comprehensive understanding of amyloid ion channels. Our Specific Aims are 1) Image 3D structure of synthetic as well as tissue-derived amyloidogenic peptides and peptides with site-specific amino acid (AA) substitutions reconstituted in lipid membrane; 2) Image open-closed conformations, in response to pharmacologic agents, of channels made of peptides, normal as well as with site-specific amino acid (AA) substitutions, 3) Correlate open-close channel conformations with ion conductance using integrated ion conductance AFM and pharmacologic agents and site-specific AA substitutions, and 4) Examine the cellular effects (e.g., calcium uptake and degeneration) of amyloid peptides. Our study will define specific amyloid structures underlying Alzheimer's and other degenerative pathophysiology and will identify specific structural motif(s) in amyloid ion channels that can then be used for designing pharmacological intervention of therapeutic value.