MOLECULAR FUNCTION OF SYNUCLEIN

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DESCRIPTION (From the applicant's abstract): The long-term goal of this project is to define the normal and pathological roles of the synucleins, and in particular alpha-synuclein. Alpha-synuclein has emerged as a major focus of investigation because of an apparent (but poorly understood) role both in neurodegenerative disease and in normal synaptic plasticity and learning. The first aim for the next project period is to complete a formal biophysical characterization of human alpha-synuclein (halphaS), focusing on its ability to interact with membrane lipids and to self-associate. This aim will be conducted in collaboration with Dr. Seelig of the University of Basel. A second aim is to compare the properties of mutant synucleins linked to the development of Parkinson's disease, as well as a non-human (canary) alpha-synuclein, to gain insight into which features are specifically conserved and which may be more likely related to pathology. A third aim is to use site-directed mutagenesis to construct mutant forms of recombinant halphaS with altered lipid binding properties. This will provide insight into the functional organization of the sequence, and will generate mutant constructs potentially useful for probing synuclein's cellular functions. A fourth aim is to map the sites upon which synuclein is phosphorylated by the protein Casein Kinase II, and to investigate the structural and functional consequences of this modification on lipid binding and phospholipase D2 inhibition. A fifth aim will employ the various recombinant constructs produced and characterized in Aims 1-4, to probe the mechanisms by which synuclein can exert effects on cell function. In collaboration with Dr. Hyman of the Massachusetts General Hospital, the hypothesis that lipid binding is necessary for cell membrane association will be formally tested, and the necessity of lipid binding for both presynaptic terminal localization and PLD2 inhibition will also be tested. Collectively, these experiments test the hypothesis that synuclein's essential molecular function is related to its conserved structural features, which allow it to bind reversibly with intracellular membranes. Manipulation of these interactions could have uses in the development of therapies for age-related diseases including Alzheimer's and Parkinson's diseases.