Protein Self-Assembly into Nanoaggregates

描述（由申请人提供）：蛋白质和肽自组装成各种大小和形态的纳米聚集体是一种广泛存在的现象，报道了大量蛋白质和合成肽。蛋白质的错误折叠和聚集与一系列被称为蛋白质错误折叠或沉积的神经退行性疾病（如阿尔茨海默病、帕金森病和亨廷顿病）的人类病理有关。尽管蛋白质错误折叠和自发聚集对疾病病理有重要贡献，但对这些过程的分子机制知之甚少。已有研究表明，长纤维的寡聚物种具有神经毒性，但这些物种的性质尚不清楚。本应用程序的目的是表征在自组装的早期阶段形成的纳米低聚物，并确定在聚集中关键参与的物种。低聚物是在聚集过程中短暂形成的，它们的表征需要能够探测此类物种的特殊方法。这个应用程序提出了一套这样的方法来实现我们的目标。基于初步数据，中心假设是二聚体的形成是促进聚集的关键步骤，二聚体复合物的寿命决定了错误折叠聚集路径。提出的研究的基本原理是，了解蛋白质错误折叠和聚集的基本机制有可能转化为控制聚集过程的具体方法。这些进展将导致新的和创新的预防和治疗蛋白质错误折叠疾病的发展。在强有力的初步数据的指导下，我们的主要假设将通过追求三个具体目标来验证：1)确定聚集动力学的关键核；2)直接对自组装过程的早期阶段进行成像；3)开发用于AFM实验的新型连接器和系索。在第一个目标下，将进行低聚态的表征。一种新的纳米方法能够探测瞬态形成的低聚物。在第二个目标下，从二聚体开始的各种大小的低聚物的组装将直接可视化。我们将能够直接测量每个低聚物浓度随时间的变化，并确定哪些物种在聚集体的生长中起着构建块的作用。据推测，纤维化需要形成一定大小的低聚物。我们将能够直接检验这个假设。将开发一种新的纳米成像方法，能够根据其大小对每种低聚物进行明确的可视化，并对其形成动力学进行定量分析。在第三个目标下，将开发一种新型聚合物连接剂，以促进前一个目标的完成。这种方法是创新的，因为它提出了一种蛋白质聚集过程的新模型，并开发了一套具有广泛生物医学应用的新型纳米技术方法来测试该模型。这项拟议中的研究意义重大，因为这些发现将为在早期阶段开发有效治疗蛋白质错误折叠疾病的方法奠定基础。

DESCRIPTION (provided by applicant): Self-assembly of proteins and peptides into nanoaggregates of various sizes and morphologies is a widespread phenomenon reported for a large number of proteins and synthetic peptides. Misfolding and aggregation of proteins are associated with a wide range of human pathologies termed protein misfolding or deposition neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. Despite the important contribution of protein misfolding and spontaneous aggregation to disease pathology, very little is known about the molecular mechanisms underlying these processes. It has been shown that oligomeric species rather long filaments are neurotoxic, but the nature of these species remains unknown. The objective of this application is to characterize the nano-oligomers formed at the early stages of self-assembly and identify the species critically involved in aggregation. The oligomers are formed transiently during the aggregation process, and their characterization requires special approaches capable of probing such species. This application proposes a set of such approaches to attain our objective. Based on preliminary data, the central hypothesis is that formation of dimers is the key step for promoting aggregation and that the lifetimes of dimeric complexes define the misfolding-aggregation paths. The rationale for the proposed research is that understanding fundamental mechanisms of protein misfolding and aggregation has the potential to translate into specific approaches to control the aggregation process. These advances will lead to the development of new and innovative preventions and treatments of protein misfolding diseases. Guided by strong preliminary data, our major hypothesis will be tested by pursuing three specific aims: 1) Identify key nuclei for the aggregation kinetic; 2) Image directly early stages of the self-assembly process; and 3) Develop novel linkers and tethers for AFM experiments. Under the first aim, the characterization of oligomeric states will be performed. A novel nanoapproach capable of probing transiently formed oligomeric species will be developed. Under the second aim, the assembly of oligomers of each size starting from dimers will be visualized directly. We will be able to measure directly the time-dependent change of the concentration of each oligomer and to identify which species play a role of building blocks for the growth of aggregates. It is hypothesized that the fibrilization requires formation of oligomers of a defined size. We will be able to directly test this hypothesis. A novel nanoimaging approach capable of unambiguous visualizing each oligomer according to its size and quantitative analysis of the kinetics of their formation will be developed. Under the third aim, a novel type of polymer linker will be developed to facilitate completion of the previous aims. The approach is innovative, because it presents a novel model for the protein aggregation process and develops a set of novel nanotechnology methods with broad biomedical applications to test this model. The proposed research is significant because the findings will lay the foundation for developing efficient treatments of protein misfolding diseases at the very early stages.