Conformations and dynamics of amyloid-induced membrane disruption

描述(申请人提供)：淀粉样纤维是一种非常稳定的自组装细丝，几乎可以由任何蛋白质形成。由于多肽很容易发生突变和/或衍生，这些特性使淀粉样蛋白成为开发生物纳米结构的一个有吸引力的系统。纤维的形成涉及到不同的中间物种的采样，包括构象和低聚物，作为组装途径的中心部分。重要的是，正是这些中间产物，而不是纤维本身，与阿尔茨海默氏症等疾病的细胞毒性有关。 从淀粉样蛋白与界面的相互作用，特别是脂质双层的相互作用，已经推断出对纤化的分子基础的有希望的见解。它们稳定和组织结构中间体，从而催化纤维的形成。也正是这些相互作用被认为是淀粉样蛋白疾病中细胞死亡的原因。后者的机制包括破坏细胞膜和消除正常细胞功能所需的离子梯度。尽管人们对膜结合的淀粉样蛋白中间体很感兴趣，而且它们与病理学有明显的相关性，但由于它们的异质性和瞬变性质，它们的分子特征一直难以捉摸。 单分子荧光测量是唯一能够量化不同的和动态的分子群体的方法。这些将被开发并应用于胰岛淀粉样多肽(IAPP)与合成脂泡的相互作用的研究。具体涉及两个方面：(1)IAPP引起膜完整性丧失的机制的表征和(2)IAPP状态和与膜破坏相关的动力学的确定。这些研究结果将提供与膜通透性有关的分子物种的描述，并深入了解纤维组装的机制。这样的理解对于开发操纵和控制纤维组装的过程至关重要，将为研究膜蛋白和淀粉样蛋白中间体的体外和体内行为提供新的工具，并最终将为确定适合于治疗学发展的新靶点提供基础。要了解蛋白质形成病理性淀粉样纤维的过程，需要详细描述在纤化过程中填充的不同分子种类。这包括膜相关结构，这些结构被认为是淀粉样蛋白疾病中细胞死亡的原因。这项工作的目的是发展单分子方法来研究淀粉样蛋白、胰岛淀粉样多肽和脂质双层之间的相互作用，以期确定适合于治疗发展的新靶点。

DESCRIPTION (provided by applicant): Amyloid fibers are remarkably stable, self-assembled filaments that can be formed from virtually any protein. As polypeptides are readily mutated and/or derivatized, these properties make amyloid an attractive system for the development of biological nano-structures. Fiber formation involves the sampling of heterogeneous intermediate species, both conformational and oligomeric, as a central part of the pathway for assembly. Importantly, it is these intermediates, not fibers themselves that are associated with cytotoxicity in diseases such as Alzheimer's. Promising insights into the molecular basis of fibrillization have been deduced from the interactions of amyloidogenic proteins with interfaces, particularly lipid bilayers. These stabilize and organize structural intermediates thereby catalyzing fiber formation. It is also these interactions that are implicated as causal to cell death in amyloid diseases. The mechanism of the latter involves disruption of membranes and elimination of the ion gradients required for normal cellular function. Despite great interest in membrane bound amyloid intermediates, and their obvious relevance to pathology, their molecular characterization has been elusive due to their heterogeneous and transient nature. Single molecule fluorescence measurements are uniquely capable of quantification of heterogeneous and dynamic molecular populations. These will be developed and applied to the study of interactions of islet amyloid polypeptide (IAPP) with synthetic lipid vesicles. Two areas are specifically addressed: (1) characterization of the mechanism of membrane integrity loss induced by IAPP and (2) determination of IAPP states and dynamics associated with membrane disruption. The results of these investigations will provide a description of the molecular species involved in membrane permeabilization, and give insight into the mechanism of fiber assembly. Such understanding is critical to developing processes for manipulation and control of fiber assembly, will provide new tools for studying the behavior of membrane proteins and amyloid intermediates both in vitro and in vivo, and finally, will provide a basis for identifying novel targets suitable for the development of therapeutics. Understanding the processes by which proteins form pathological amyloid fibers requires a detailed description of the heterogeneous molecular species populated during fibrillization. This includes the membrane-associated structures that are implicated as causal agents in cell death in amyloid diseases. The aim of this work is to develop single molecule methods for studying interactions between the amyloidogenic protein, islet amyloid polypeptide, and lipid bilayers with a view towards identifying novel targets suitable for the development of therapeutics.