Amyloid ion channels to design therapeutics for neurodegenerative diseases

描述（由申请人提供）：异常（或错误）折叠改变蛋白质的三维构象，从天然（可溶形式）到非天然（不可溶形式）多态淀粉样蛋白结构。蛋白质错误折叠与神经退行性疾病（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）、全身性疾病（II型糖尿病、轻链淀粉样变性相关癌症）和其他疾病（囊性纤维化）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细胞病理生理反应。了解蛋白质错误折叠和由此产生的三维构象诱导病理生理活动一直是一个重要但具有挑战性的研究领域。淀粉样纤维的形成及其预防机制正在被广泛研究，尽管淀粉样纤维并不直接导致神经退行性疾病；最近的研究表明，只有球状淀粉样蛋白才足以引起病理生理反应。球状低聚物介导毒性的最直接机制可能涉及其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的球状淀粉样蛋白形成离子通道破坏细胞离子稳态造成的。因此，调节其通道结构和活动的分子和其他干预措施可以用于有效的治疗。事实上，淀粉样蛋白肽在天然细胞和人工膜中都能诱导离子电导。膜结合淀粉样蛋白复合物的结构研究一直受到限制。我们的研究表明，与几种疾病相关的淀粉样肽形成多态离子通道。这项持续的建议主要集中在与阿尔茨海默病（AD）相关的淀粉样蛋白β (Ab)肽形成毒性通道。我们打算确定Ab肽的三维结构多态性，并确定Ab肽的氨基酸（AA）表位，然后可以作为设计有效治疗AD的靶点。我们将继续使用AFM成像、离子电导记录、分子动力学（MD）模拟和细胞钙摄取和变性等多维和互补的方法，以获得对淀粉样蛋白离子通道的全面了解。我们的具体目标是：1)合成和组织来源的淀粉样蛋白肽和在脂膜中重组的具有位点特异性氨基酸（AA）取代的肽的图像三维结构；2)在药物作用下，成像由多肽组成的通道的开闭构象，包括正常的和位点特异性氨基酸（AA）取代的；3)使用离子电导综合原子力显微镜（AFM）、药物作用剂和位点特异性氨基酸取代，将开闭通道构象与离子电导联系起来；4)检查淀粉样肽的细胞效应（如钙摄取和变性）。我们的研究将确定阿尔茨海默病和其他退行性病理生理的特定淀粉样蛋白结构，并将确定淀粉样蛋白离子通道中的特定结构基元，然后可用于设计具有治疗价值的药物干预。

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.