To understand the molecular basis of any biological process, it is critical that one is not only able to visualize and monitor molecular events that underlie this process, but also to possess the tools to manipulate these events in a spatial and temporal fashion both in and out of the cell. The overall objective of this proposal is to apply chemical biology approaches to allow real time monitoring of protein aggregation and to dissect the role of specific disease-associated post-translational modifications, phosphorylation, nitration, and truncation on the structure, aggregation, and biochemical properties of monomeric a-syn in health and disease. To achieve these goals, we plan to use a combination of organic chemistry, molecular biology, proteomics, protein engineering, and semisynthetic strategies to facilitate site-specific introduction of post-translational modifications that can be masked and activated in a controllable manner, both inside and outside living cells. Modified synthetic ±-syn will be introduced into primary neurons and cellular models of synucleinopathies and the consequences of masking or activating specific modifications will be assessed using biochemical, immunofluorescence, and live imaging techniques (Specific Aim 1). The absence of specific molecular probes that allow in vivo monitoring and quantitative measurement of toxic misfolded and aggregation intermediates represents a major impediment to understanding the relationship among protein misfolding, post-translational modification, protein aggregation, neurodegeneration, and cell death in PD and other neurodegenerative disorders. To address this challenge, we plan to develop and characterize novel antibodies that target different species along the amyloid formation pathway of ±-syn (Specific Aim 2).
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