Identification of Novel Alpha Synuclein Binding Protein

新型α突触核蛋白结合蛋白的鉴定

• 批准号: 6721561
• 负责人: BRETT P LAURING
• 金额: $ 37.81万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6721561
• 关键词: Parkinson' s disease; alpha synuclein; binding proteins; calmodulin; conformation; crosslink; fluorescence resonance energy transfer; molecular assembly /self assembly; mutant; protein folding; protein protein interaction; protein purification; protein structure function; structural biology; technology /technique development

DESCRIPTION (provided by applicant): Parkinson's Disease (PD) is the second most common neurodegenerative disease. As in many other neurodegenerative diseases, conformational alteration of a specific neuronal protein results in the accumulation of fibrillar amyloid inclusions, which in the case of Parkinson's disease, are termed Lewy Bodies (LBs). LBs have a fibrillar core with the fibrils being comprised primarily of a protein of unknown function called alpha-synuclein. Alpha-synuclein mutations cause autosomal dominant Parkinson's disease. Thus both human genetic and histologic evidence link synuclein to Parkinson's disease. Alpha-synuclein is a 140 aa protein which is 'natively unfolded' meaning that it has no identifiable secondary structure. However, in the presence of certain lipid membranes is can fold into a alpha helical conformation, and when incubated alone can fold into a beta-sheet rich conformation which allows it to form amyloid fibrils resembling those seen in Lewy bodies. Consistent with the hypothesis that alteration of synuclein conformation is linked to development of Parkinson's disease, purified mutant synuclein fibrillizes more rapidly than wild-type protein in vitro. Overexpression of synuclein as a transgene results in formation of Lewy body-like pathology in mice and flies. Synuclein expressed at endogenous levels rarely forms amyloid (only in PD patients), is not stably membrane-associated, and remains 'unfolded'. The discrepancy between the in vivo folding parameters and those observed in vitro leads us to hypothesize that synuclein-interacting molecules may regulate synuclein conformation, stabilize it in the 'unfolded' state, or regulate membrane binding. We therefore set up a novel photo-cross linking assay heretofore not used to study synuclein to identify synuclein binding proteins present in brain extracts and present at endogenous levels of expression to begin to determine how synuclein conformation is regulated. We have identified novel synuclein binders. We propose to develop a fluorescence resonance energy transfer assay capable of indicating synuclein conformation both in vivo and in vitro. That will allow for screening of proteins and synthetic agents capable of altering synuclein aggregation. These studies will enable us to define the range of proteins or agents to be further characterized in in vivo models of Parkinson's disease.

描述(申请人提供)：帕金森病(PD)是第二种最常见的神经退行性疾病。与许多其他神经退行性疾病一样，特定神经元蛋白的构象改变会导致纤维淀粉样包涵体的积累，在帕金森病的情况下，这被称为路易小体(Lbs)。LBS有一个纤维核心，纤维主要由一种功能未知的蛋白质组成，称为α-突触核蛋白。α-突触核蛋白突变导致常染色体显性遗传性帕金森氏病。因此，人类遗传和组织学证据都将突触核蛋白与帕金森氏症联系起来。α-突触核蛋白是一种140个氨基酸的蛋白质，是“天然未折叠的”，这意味着它没有可识别的二级结构。然而，在某些脂膜的存在下，它可以折叠成α螺旋构象，当单独孵育时，它可以折叠成丰富的β-折叠构象，这使得它能够形成类似于路易小体的淀粉样纤维。与突触核蛋白构象改变与帕金森病的发生有关的假设一致，纯化突变体突变体突触核蛋白在体外比野生型蛋白更快地形成纤维。转基因突触核蛋白的过表达可导致小鼠和果蝇形成路易体样病理。 在内源性水平表达的突触核蛋白很少形成淀粉样蛋白(仅在帕金森病患者中)，不稳定地与膜相关，并且保持未折叠。体内折叠参数与体外观察到的折叠参数之间的差异使我们假设，突触核蛋白相互作用的分子可能调节突触核蛋白的构象，使其稳定在未折叠状态，或调节膜结合。因此，我们建立了一种到目前为止还没有用于研究突触核蛋白的新型光交联试验，以鉴定存在于脑提取液中和存在于内源表达水平的突触核蛋白结合蛋白，以开始确定突触核蛋白构象是如何调控的。我们已经确定了新的突触核蛋白结合子。我们建议建立一种能够在体内和体外显示突触核蛋白构象的荧光共振能量转移分析方法。这将使筛选能够改变突触核蛋白聚集的蛋白质和合成试剂成为可能。这些研究将使我们能够确定在帕金森病的活体模型中进一步表征的蛋白质或制剂的范围。