Protein Self Assembly into Nanoaggregates

 描述（由申请人提供）：淀粉样蛋白的聚集与称为蛋白质错误折叠或沉积神经退行性疾病的多种人类病理学有关，包括阿尔茨海默病、帕金森病和亨廷顿病。已经显示淀粉样蛋白聚集体的寡聚物种类是神经毒性的。然而，这些物种的性质仍然未知。尽管低聚物种类在毒性方面以及在正常生理事件中具有重要性，但关于蛋白质自组装的分子机制的知识非常有限。本申请的目的是在一定水平上表征每种低聚物，使我们能够理解纳米组装过程的分子机制。然而，低聚物瞬时形成的事实基本上阻碍了它们的表征。为了克服这一障碍，我们提出了一种新的方法，在该方法中，将限定尺寸的寡聚体组装在基于聚合物的柔性纳米阵列（FNA）上，这将使得能够使用各种方法对其进行表征。基于在当前资助期间获得的数据，我们假设自组装是由增加的、大小依赖的、分子间相互作用和低聚物的稳定性驱动的。为了验证这一假设，我们将通过应用一组单分子方法，结合详细的计算分析，彻底表征FNA组装的寡聚体。在强有力的初步数据的指导下，我们将通过以下三个具体目标来阐述我们的主要假设：目标1）开发一种新的灵活的纳米阵列方法来测量低聚物内的相互作用;目标2）使用一种新的拴系方法直接测量低聚物的寿命;目标3）使用尖端增强拉曼光谱（TERS）方法展示单个聚集淀粉样蛋白的二级结构分析。提出这些目标的理由是，了解蛋白质错误折叠和聚集的基本机制具有很强的潜力，可以转化为控制聚集过程的具体方法。这些进展预计将导致开发新的和创新的预防策略和治疗蛋白质错误折叠疾病，如阿尔茨海默病。该应用是创新的，因为它为蛋白质聚集现象提供了一种新的方法，并开发了一套具有广泛生物医学应用的新纳米技术方法。这项研究意义重大，因为这些发现将为在早期阶段有效治疗蛋白质错误折叠疾病奠定基础。此外，选择大小的寡聚体组装成FNA的可用性开辟了它们作为诊断工具如免疫测定开发中的靶标的前景。此外，考虑到低聚物，而不是包括原纤维的较大聚集体，被认为是神经毒性物质，具有所需大小的低聚物的可用性将为开发针对阿尔茨海默病、帕金森病和其他神经退行性疾病的有效免疫预防、诊断和治疗策略打开现实前景。

 DESCRIPTION (provided by applicant): Aggregation of amyloid proteins is associated with a wide range of human pathologies termed protein misfolding or deposition neurodegenerative disorders, which include Alzheimer's, Parkinson's, and Huntington's diseases. It has been shown that oligomeric species of amyloid aggregates are neurotoxic. Still, the nature of these species remains unknown. Despite this importance of oligomeric species with respect to toxicity as well as in normal physiological events, knowledge regarding the molecular mechanisms underlying proteins self-assembly is very limited. The objective of this application is to characterize each oligomer at a level that will allow us to understand the molecular mechanism of the nanoassembly process. However, the fact that oligomers are formed transiently essentially impedes their characterization. To overcome this obstacle, we propose a novel approach in which oligomers of a defined size are assembled on a polymer- based Flexible Nanoarray (FNA), which will enable the use of various methods for their characterization. Based on data obtained during the current funding period, we hypothesize that the self-assembly is driven by increased, size-dependent, intermolecular interaction and stability of the oligomers. To test this hypothesis, we will thoroughly characterize FNA-assembled oligomers by applying a set of single-molecule approaches, combined with detailed computational analyses. Guided by strong preliminary data, we will text our major hypothesis through the following three specific aims: Aim 1) Develop a novel flexible nanoarray approach to measure interactions within oligomers; Aim 2) Directly measure directly the lifetimes of oligomers using a novel, tethered approach; and Aim 3) Demonstrate secondary structural analysis for individual aggregated amyloids using a Tip-Enhanced Raman Spectroscopy (TERS) approach. The rationale for the proposed aims is that understanding fundamental mechanisms of protein misfolding and aggregation has the strong potential to translate into specific approaches to control the aggregation process. These advances are expected to lead to the development of new and innovative preventative strategies and treatments for protein misfolding diseases like Alzheimer's disease. The application is innovative, because it presents a novel approach to the protein aggregation phenomenon and develops a set of new nanotechnology methods with broad biomedical applications. The proposed research is significant because the findings will lay the foundation for efficient treatments against protein misfolding diseases at the very early stages. Additionally, the availability of oligomers of select sizes assembled as FNAs opens prospects for their use as targets in the development of diagnostic tools such as immunoassays. Moreover, given that oligomers, rather than larger aggregates including fibrils, are considered neurotoxic species, the availability of oligomers with desired sizes will open realistic prospects for the development of efficient immunological preventive, diagnostic, and therapeutic strategies for Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders.