Semi-synthetic a-Synuclein for Tracking Aggregation and Cell-to-Cell Transmission

描述(由申请人提供)：保持蛋白质的稳定、折叠状态对于正常的细胞功能是必不可少的。蛋白质错误折叠是至少11种神经退行性疾病的基础，包括路易体痴呆和帕金森病。目前帕金森氏症的治疗只提供症状缓解，并观察到显著的副作用。逆转或阻断聚集的药物，结合早期诊断，为保存患者记忆的治愈提供了最好的前景。要设计这样的药物，必须了解聚集和传播的过程。我们建议使用我们实验室开发的新的荧光技术来从结构上表征α-突触核蛋白(αS)的聚集体，这是帕金森氏病的病理标志。αS单体错误折叠，形成几个单体单元(低聚物)的小聚集体，然后形成长纤维，可能会产生对神经元有毒的难以溶解的缠结。确定这些低聚物的分子结构对于阐明参与纤维形成的聚集途径是至关重要的。此外，αS跨膜行为的结构特征可能有助于解释病理从神经元到神经元的传播。然而，实验和理论工作由于化学计量不均一性、非结构化区域的存在以及低聚物的时间不稳定性而变得复杂。我们最近开发的硫代酰胺荧光猝灭作为一种依赖时间的结构测定方法，为淀粉样蛋白的表征提供了一个强有力的新工具。由于硫代酰胺可以插入到肽骨架的任何位置，它们可以提供分子内和分子间接触的最小扰动、单一残基探针。荧光技术非常适合于解决齐聚物结构测定的三个突出问题：异质性(因为可以使用单分子技术)、样品环境(因为研究可以在细胞培养的稀释条件下进行)和时间不稳定性(可以实时监测变化)。利用硫代酰胺荧光猝灭，我们将生成聚集中间体的结构模型，并通过在培养细胞中进行的荧光测量来验证这些模型。

DESCRIPTION (provided by applicant): The maintenance of proteins in their stable, folded state is essential to proper cellular function. Protein misfolding underlies at least 11 neurodegenerative disorders, including Lewy Body dementia and Parkinson's disease. Current Parkinson's disease treatments provide only symptom relief, and significant side effects are observed. Drugs that reverse or block aggregation, combined with early diagnosis, provide the best prospect for a cure that preserves the patient's memories. To design such drugs, one must understand the process of aggregation and propagation. We propose to use novel fluorescence techniques developed in our laboratory to structurally characterize aggregates of the protein α-synuclein (αS), the pathological hallmark of Parkinson's disease. αS monomers misfold, forming small aggregates of a few monomer units (oligomers) before going on to form long fibrils that can create insoluble tangles that are toxic to neurons. Determining the molecular structure of these oligomers is crucial for elucidating the aggregation pathway involved in fibril formation. Furthermore, structural characterization of the membrane-crossing behavior of αS may help to explain the spread of pathology from neuron-to-neuron. However, experimental and theoretical efforts are complicated by stoichiometric heterogeneity, the presence of unstructured regions, and the temporal instability of oligomers. Our recent development of thioamide fluorescence quenching as a time-dependent structure determination method offers a powerful new tool to be applied to amyloid characterization. Since thioamides can be inserted at any position in the peptide backbone, they can provide minimally-perturbing, single residue probes of intra- and intermolecular contacts. Fluorescence techniques are well suited to address three of the outstanding problems with oligomer structure determination: heterogeneity (because single-molecule techniques can be used), sample environment (because studies can be carried out under dilute conditions in cell cultures), and temporal instability (changes can be monitored in real time). Using thioamide fluorescence quenching, we will generate structural models of the aggregation intermediates and validate these models with fluorescence measurements made in cultured cells.