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

描述（由申请人提供）：蛋白质维持其稳定折叠状态对正常细胞功能至关重要。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仅提供症状缓解，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繁殖的过程。我们建议使用我们实验室开发的新型荧光技术来表征蛋白质α-突触核蛋白（αS）的聚集体的结构，α S是帕金森病的病理标志。αS单体错误折叠，形成一些单体单元（寡聚体）的小聚集体，然后形成长纤维，可以产生对神经元有毒的不溶性缠结。确定这些低聚物的分子结构对于阐明原纤维形成中涉及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，实验和理论的努力是复杂的化学计量异质性，非结构化区域的存在下，和低聚物的时间不稳定性。我们最近开发的硫代酰胺荧光猝灭作为一种时间依赖性的结构测定方法提供了一个强大的新工具，适用于淀粉样蛋白的表征。由于硫代酰胺可以插入肽骨架中的任何位置，因此它们可以提供分子内和分子间接触的最小干扰的单残基探针。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（因为可以在细胞培养物中的稀释条件下进行研究）和时间不稳定性（可以在真实的时间内监测变化）。使用硫代酰胺荧光猝灭，我们将生成聚集中间体的结构模型，并验证这些模型与培养细胞中的荧光测量。

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.