Interfacially activated aggregation of alpha-synuclein

 描述（由申请人提供）：β-突触核蛋白的聚集和纤维化与帕金森病的进展有关，目前帕金森病还没有治愈方法。本研究计划的重点是β-突触核蛋白聚集和纤维化的分子起始步骤。 该项目建立在PI的发现基础上，即β-突触核蛋白似乎对聚集有抵抗力，除非暴露于非流体疏水界面或预先形成的种子。该研究使用化学官能化的二氧化硅基底来测量界面处的β-突触核蛋白构象，并使用相同官能化的混合球来测定界面对聚集和纤维化的影响。该平台允许系统评估 界面的物理和化学性质，β-突触核蛋白的构象变化，以及聚集和纤维化的起始之间的关系。本研究将确定界面的物理和化学性质以及导致聚集和纤维化的β-突触核蛋白结构的变化。 然后，该项目建立在对聚集和纤维化的第一步的鉴定的基础上，开发了一种荧光测定法，用于测定在第一步中发生的β-突触核蛋白的变化。该测定旨在实现单分子灵敏度。该测定将用于了解更多关于β-突触核蛋白聚集和fifilization的第一步。 为了评估β-突触核蛋白在没有成核配偶体的情况下抵抗纤维化的假设，将评估不含成核界面的样品以试图找到不包括成核配偶体的纤维化条件。 最后，开发了荧光测定法来探测导致絮凝的聚集的第一步 将用于评估大分子结合伴侣。将使用该测定根据它们在β-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维化来对大分子进行分类。这些测定法将评价其作为细胞裂解物中β-突触核蛋白结合配偶体的发现试剂的未来效用。

 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.