Synuclein Binding Proteins: Conformational Regulators

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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.