Synuclein Binding Proteins: Conformational Regulators

突触核蛋白结合蛋白：构象调节剂

• 批准号: 6573233
• 负责人: BRETT P LAURING
• 金额: $ 19.42万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6573233
• 关键词: HeLa cells; alpha synuclein; amyloid proteins; binding sites; calmodulin; conformation; crosslink; fluorescence resonance energy transfer; gene mutation; protein folding; protein protein interaction; protein purification; protein structure; transfection

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 PD, are termed Lewy Bodies (LBs). LBs have a fibrillar core with the fibrils being comprised primarily of a protein of unknown function called a synuclein. A synuclein mutations cause autosomal dominant PD. Thus both human genetic and histologic evidence link synuclein to PD. a synuclein is a 140 as 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 helical conformation, and when incubated alone can fold into a 0-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 PD, purified mutant synuclein fibrillizes more rapidly than wild-type protein in vitro. Over expression 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 is the "unfolded" state, or regulate membrane binding. We therefore set up a novel photocrosslinking 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 vivo models of PD.

描述（由申请人提供）： 帕金森病（Parkinson's Disease，PD）是第二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导致纤维状淀粉样蛋白内含物的积累，在PD的情况下，其被称为路易体（LB）。LB具有纤维状核心，其中纤维主要由称为突触核蛋白的未知功能的蛋白质组成。突触核蛋白突变导致常染色体显性PD。因此，人类遗传学和组织学证据都将突触核蛋白与PD联系起来。突触核蛋白是“天然未折叠”的蛋白质，这意味着它没有可识别的二级结构。然而，在某些脂质膜的存在下，它可以折叠成螺旋构象，并且当单独孵育时可以折叠成富含0-片层的构象，这允许它形成类似于路易体中所见的淀粉样蛋白原纤维。与突触核蛋白构象的改变与PD的发展相关的假设一致，纯化的突变体突触核蛋白在体外比野生型蛋白质更快地flipzes。作为转基因的突触核蛋白的过度表达导致小鼠和果蝇中形成路易体样病理。 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PD患者中），不稳定地与膜结合，并且保持“未折叠”。体内折叠参数和体外观察到的参数之间的差异使我们假设突触核蛋白相互作用分子可能调节突触核蛋白构象，稳定它的“未折叠”状态，或调节膜结合。因此，我们建立了一种新的光交联测定法，迄今尚未用于研究突触核蛋白，以鉴定脑提取物中存在的突触核蛋白结合蛋白，并以内源性表达水平存在，从而开始确定突触核蛋白构象是如何调节的。我们已经确定了新的突触核蛋白粘合剂。我们建议开发一种荧光共振能量转移测定法，能够指示突触核蛋白的构象在体内和体外。这将允许筛选能够改变突触核蛋白聚集的蛋白质和合成剂。这些研究将使我们能够确定在PD体内模型中进一步表征的蛋白质或试剂的范围。