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: 7155547
• 负责人: MICHAEL L SHELANSKI
• 金额: $ 35.85万
• 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 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155547
• 关键词: Acetylcysteine; Adopted; Amino Acid Motifs; Amphipathic Alpha Helix; Amyloid; Amyloid Fibrils; Behavior; Binding; Binding Proteins; Binding Sites; Biological Assay; Brain; Calmodulin; Cells; Cytosol; Data; Development; Energy Transfer; Fluorescence Resonance Energy Transfer; Helix (Snails); Histologic; Human; Human Genetics; In Vitro; Incubated; Lewy Bodies; Link; Lipid Bilayers; Lipoproteins; Maps; Membrane; Membrane Lipids; Molecular Conformation; Mus; Mutation; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenesis; Pathology; Patients; Protein Overexpression; Proteins; Range; Rate; Recombinants; Screening procedure; Structure; Testing; Thinking; Time; Transgenes; alpha synuclein; base; beta-sheet-rich conformation; conformational alteration; crosslink; ear helix; fly; in vivo; in vivo Model; mutant; novel; small molecule; synthetic protein; synuclein; therapeutic target

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（由申请人提供）：帕金森氏病（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会导致原纤维淀粉样蛋白夹杂物的积累，在帕金森氏病的情况下，这种蛋白质被称为Lewy Bodies（LBS）。 LB具有原纤维核，其原纤维主要由称为α-核蛋白的未知功能的蛋白质组成。 α-突触核蛋白突变引起常染色体显性帕金森氏病。因此，人类遗传和组织学证据都将突触核蛋白与帕金森氏病联系起来。 α-突触核蛋白是一种140 AA蛋白，“本地展开”意味着它没有可识别的二级结构。但是，在某些脂质膜的存在下，可以折叠成α螺旋构象，并且单独孵育时可以折叠成浓beta型构象，从而使其可以形成类似于Lewy身体的淀粉样蛋白原纤维。与假设的假设是，突触核蛋白构象的改变与帕金森氏病的发展有关，纯化突变体突触蛋白在体外比野生型蛋白更快地纤维化。突触核蛋白作为转基因的过表达导致小鼠和苍蝇形成Lewy身体样病理学。 在内源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），与膜相关，并且保持“展开”。体内折叠参数与观察到的体外观察到的差异导致我们假设突触核蛋白相互作用的分子可能调节脊髓灰质素构象，在“展开的”状态下稳定它，或者调节膜结合。因此，我们建立了一种新型的照片交叉连接测定，迄今未用于研究突触核蛋白以鉴定出存在于脑提取物中的突触核蛋白结合蛋白，并存在于内源性水平的表达水平，以开始确定如何调节突触核蛋白构象。我们已经确定了新型突触核蛋白粘合剂。我们建议开发一种能够在体内和体外表明突触核蛋白构象的荧光共振能量转移测定法。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在帕金森氏病体内模型中定义蛋白质或剂的范围。