Interfacially activated aggregation of alpha-synuclein

 描述(由申请人提供)：-突触核蛋白的聚集和纤化与帕金森病的进展有关，目前尚无治愈方法。本研究的重点是-突触核蛋白聚集和纤化的分子启动步骤。该项目建立在PI发现-突触核蛋白似乎抵抗聚集的基础上，除非暴露在非流体疏水界面或预制种子中。该研究使用化学功能化的二氧化硅底物来测量界面上的-突触核蛋白构象，并使用相同官能化的混合球来测试界面对聚集和纤化的影响。该平台允许进行系统评估 研究了界面的物理和化学性质、-突触核蛋白的构象变化与聚集和纤化的启动之间的关系。这项研究将确定界面的物理和化学性质以及导致聚集和纤化的-突触核蛋白结构的变化。然后，该项目在确定第一步聚集和纤化的基础上，开发一种荧光分析方法，用于第一步中发生的-突触核蛋白的变化(S)。该检测旨在实现单分子敏感性。该分析将被用来更多地了解-突触核蛋白聚集和纤化的第一步。为了评估-突触核蛋白在没有成核伙伴的情况下抵抗纤化的假设，将对没有成核界面的样品进行评估，以试图找到不包括成核伙伴的成核条件。最后，发展了荧光分析来探测导致纤化的聚集的第一步。 将用于评估大分子结合伙伴。这些大分子将根据它们在-突触核蛋白中诱导的结构类型以及它们如何抑制或促进聚集和纤化来使用该分析进行分类。这些分析将评估它们作为细胞裂解物中-突触核蛋白结合伙伴的发现试剂的未来用途。

 DESCRIPTION (provided by applicant): Aggregation and fibrillization of -synuclein has been implicated in the progression of Parkinson's Disease, which currently has no cure. This research project is focussed on the molecular steps for initiation of -synuclein aggregation and fibrillization. The project builds on the PI's discovery that -synuclein appears resistant to aggregation except when exposed to non-fluid hydrophobic interfaces or to preformed seeds. The research uses chemically functionalized silica substrates to measure -synuclein conformation at the interface, and uses identically functionalized mixing balls to assay for the interfacial influence on aggregation and fibrillization. This platform allows systematic evaluation of the relationship between the physical and chemical properties of the interface, the conformational changes in -synuclein, and the initiation of aggregation and fibrillization. The research will identify the physical and chemical properties of interface and the changes in -synuclein structure that lead to aggregation and fibrillization. The project then builds on the identification of that first step of aggregation and fibrillization to develop a fluorescence assayfor the changes in -synuclein that occur during the first step(s). The assay is intended to enable single-molecule sensitivity. The assay will be used to learn more about the first steps of -synuclein aggregation and fibrillization. To evaluate the hypothesis that -synuclein resists fibrillization without a nucleation partner, samples free from nucleating interfaces will be evaluated to try to find conditions for fibrillization that do not include nucleating parnters. Fially the fluorescence assays developed to probe the first steps of aggregation leading to fibrillization will be used to evaluate macromolecular binding partners. The macromolecules will be classified using the assay according to the type of structure they induce in -synuclein and how they inhibit or promote aggregation and fibrillization. These assays will evaluated for their future utiity as discovery reagents for -synuclein binding partners in cell lysates.