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

描述（由申请人提供）：维持蛋白质的稳定，折叠状态对正常的细胞功能至关重要。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。目前的帕金森病治疗只提供症状缓解，并观察到明显的副作用。逆转或阻断记忆聚集的药物与早期诊断相结合，为保留患者记忆的治疗提供了最好的前景。为了设计这样的药物，人们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型荧光技术来结构表征α-突触核蛋白（αS）的聚集体，这是帕金森病的病理标志。αS单体错误折叠，在形成长原纤维之前形成少数单体单元（低聚物）的小聚集体，而长原纤维可以产生对神经元有毒的不可溶缠结。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至关重要。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，由于化学计量异质性、非结构化区域的存在和低聚物的时间不稳定性，实验和理论的努力变得复杂。我们最近开发的硫酰胺荧光猝灭作为一种随时间变化的结构测定方法，为淀粉样蛋白表征提供了一种强大的新工具。由于硫胺可以插入到肽主链的任何位置，它们可以提供最小干扰的分子内和分子间接触的单残留探针。荧光技术非常适合解决低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（变化可以实时监测）。利用硫酰胺荧光猝灭，我们将生成聚集中间体的结构模型，并在培养细胞中进行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.