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

用于跟踪聚集和细胞间传输的半合成α-突触核蛋白

基本信息

批准号：
8421217
负责人：
Ernest James Petersson
金额：
$ 30.45万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-28 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8421217
关键词：
Address Adverse effects Amino Acid Substitution Amino Acids Amyloid Amyloid Fibrils Amyloid Proteins Aromatic Amino Acids Behavior Cell Aggregation Cell Culture Techniques Cell physiology Cells Centrifugation Chemicals Crystallography Cultured Cells Development Diagnostic Early Diagnosis Electron Transport Energy Transfer Environment Fluorescein Fluorescence Fluorescence Microscopy Heterogeneity In Situ In Vitro Label Laboratories Lewy Bodies Lewy Body Disease Life Ligation Location Maintenance Measurement Membrane Membrane Proteins Memory Methods Modeling Modification Molecular Conformation Molecular Structure Monitor Mutagenesis Mutation Nervous system structure Neurodegenerative Disorders Neurofibrillary Tangles Neurons Parkinson Disease Pathology Patients Peptides Pharmaceutical Preparations Physiological Play Positioning Attribute Process Protein Region Proteins Reaction Recruitment Activity Research Role Route Sampling Site Solutions Structural Models Structure Symptoms Techniques Thioamides Time Toxic effect Vertebral column aggregation pathway alpha synuclein amyloid peptide analog design fluorophore intermolecular interaction monomer novel protein aggregate protein misfolding research study single molecule synuclein therapy design tool transmission process uptake

项目摘要

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 ?-synuclein (?S), the pathological hallmark of Parkinson's disease. ?S 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 ?S 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. PUBLIC HEALTH RELEVANCE: The aggregation of amyloid peptides into oligomers (clusters) and ultimately into fibrillar tangles called Lewy bodies plays a pivotal role in the development of Parkinson's disease. The proposed research will study the structures of intermediates in the aggregation process in vitro and in cultured cells to aid in the design of therapies targeted toward alleviating their toxicity.

描述（由申请人提供）：蛋白质维持稳定的折叠状态对于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。目前帕金森病的治疗只能缓解症状，并且观察到明显的副作用。逆转或阻止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佳前景。要设计此类药物，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型荧光技术来表征蛋白质β-突触核蛋白（？S）的聚集体，这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（低聚物）的小聚集体，然后形成长原纤维，从而形成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，?S 跨膜行为的结构特征可能有助于解释神经元之间的病理传播。然而，化学计量异质性、非结构化区域的存在以及低聚物的时间不稳定性使实验和理论工作变得复杂。我们最近开发的硫代酰胺荧光猝灭作为一种时间依赖性结构测定方法，为淀粉样蛋白表征提供了一个强大的新工具。由于硫代酰胺可以插入肽主链的任何位置，因此它们可以提供干扰最小的单残基探针，用于分子内和分子间接触。荧光技术非常适合解决低聚物结构测定中的三个突出问题：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物的稀释条件下进行）和时间不稳定性（可以实时监控变化）。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，并通过在培养细胞中进行的荧光测量来验证这些模型。公共健康相关性：淀粉样肽聚集成低聚物（簇）并最终形成称为路易体的纤维缠结，在帕金森病。拟议的研究将研究体外和培养细胞中聚集过程中中间体的结构，以帮助设计旨在减轻其毒性的疗法。