Identification of Novel Alpha Synuclein Binding Protein

描述（由申请人提供）：帕金森病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包涵体的积累，在帕金森病的情况下，称为路易体（LBs）。lb有一个纤原核，纤原主要由一种功能未知的蛋白质-突触核蛋白组成。α -突触核蛋白突变导致常染色体显性帕金森病。因此，人类遗传和组织学证据将突触核蛋白与帕金森病联系起来。α -突触核蛋白是一种140 aa的蛋白质，它是“天然展开的”，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α螺旋构象，当单独孵卵时，它可以折叠成富含β片的构象，这使得它可以形成类似于路易体中所见的淀粉样原纤维。与突触核蛋白构象的改变与帕金森病的发展有关的假设一致，纯化突变的突触核蛋白在体外比野生型蛋白更快地成纤维。突触核蛋白作为一种转基因的过度表达导致小鼠和果蝇的路易体样病理的形成。

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.