Protein Self-Assembly into Nanoaggregates

描述(申请人提供)：蛋白质和多肽自组装成不同大小和形态的纳米聚集体是大量蛋白质和合成多肽的普遍现象。蛋白质的错误折叠和聚集与广泛的人类病理有关，称为蛋白质错误折叠或沉积神经退行性疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿病。尽管蛋白质错误折叠和自发聚集在疾病病理中有重要贡献，但人们对这些过程背后的分子机制知之甚少。已有研究表明，寡聚物种相当长的细丝具有神经毒性，但这些物种的性质尚不清楚。这项应用的目的是表征在自组装的早期阶段形成的纳米低聚物，并识别与聚集密切相关的物种。低聚物是在聚集过程中瞬时形成的，它们的表征需要能够探测这种物种的特殊方法。这项申请提出了一套这样的方法来实现我们的目标。根据初步数据，中心假设是二聚体的形成是促进聚集的关键步骤，二聚体络合物的寿命决定了错误折叠-聚集路径。这项拟议研究的基本原理是，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。这些进展将导致蛋白质错误折叠疾病的新的和创新的预防和治疗的发展。在强大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)直接成像自组装过程的早期阶段；以及3)为AFM实验开发新的连接物和系链。在第一个目标下，将进行低聚态的表征。将开发一种新的纳米方法，能够探测瞬时形成的低聚物物种。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被直接可视化。我们将能够直接测量每个低聚物浓度随时间的变化，并确定哪些物种起到聚集体生长的构建块的作用。假设纤化需要形成限定大小的低聚物。我们将能够直接检验这一假设。将开发一种新的纳米成像方法，能够根据其大小明确地显示每一种低聚物，并对其形成动力学进行定量分析。在第三个目标下，将开发一种新型的聚合物连接物，以促进完成先前的目标。该方法具有创新性，因为它为蛋白质聚集过程提供了一个新的模型，并开发了一套具有广泛生物医学应用的新的纳米技术方法来测试该模型。这项拟议的研究意义重大，因为这些发现将为在非常早期阶段开发有效的蛋白质错折叠疾病治疗方法奠定基础。

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.