Studying Alpha-Synuclein Post-Translational Modifications through Novel Chemical-Biology Tools

Project Summary The goal of this proposal is to develop a modular chemical biology tool to probe the biology of alpha-synuclein post-translational modifications. Parkinson’s disease is a devastating neurodegenerative disease characterized by pathological aggregations that include alpha-synuclein (a-syn). a-syn is a small (15 kDa) intrinsically disordered protein with the propensity to aggregate into cytotoxic oligomers or fibrils. It is suspected to play a substantial role in the pathophysiology of Parkinson’s disease. a-syn has dozens of recognized post translational modifications (PTMs), including phosphorylation, acetylation, or arginylation. Prior work has demonstrated that some PTMs, such as arginylation or acetylation, are associated with reduced pathogenicity of a-syn fibrils. Phosphorylation, by contrast, is associated with a-syn pathology and its presence is treated as a proxy for a-syn-fibril-induced cytotoxicity in cell and animal models. However, prior research into a-syn PTMs has been hindered by the lack of tools to directly induce PTMs of fibrils. Without methods to selectively modulate the PTMs of a-syn fibrils it is difficult to make causal inferences on the native function of each PTM. Bifunctional molecules are one method of introducing such PTMs, as by binding to PTM enzymes and the protein of interest they can cause proximity-induced labeling of the protein of interest. Prior work from our laboratory has demonstrated the use of bifunctional molecules to induce ubiquitinylation and degradation of target proteins labeled with e. Coli Dihydrofolate Reductase (eDHFR), a small (18 kDa) protein tag targetable with trimethoprim (TMP) based bifunctional molecules. In this proposal, I will adapt the eDHFR protein tag, alongside TMP-based bifunctional molecules targeting a-syn fibrils, to create a modular system for the small- molecule-induced post translational modification of a-syn fibrils. By this, we will gain a modular, selective, and inducible way to induce PTMs onto alpha synuclein fibrils, empowering research into the role of a-syn PTMs. In aim 1 I will synthesize TMP-based bifunctional molecules targeting a-syn fibrils and eDHFR and I will use them to induce the arginylation or acetylation of a-syn fibrils in cells transduced with eDHFR-enzyme fusion proteins. I will then use this system to study the impact of arginylation or acetylation on the cytotoxicity of a-syn fibrils in a Parkinson’s disease model system. In aim 2 I will adapt the system to study the effects of the phosphorylation of a-syn fibrils in Parkinson’s models. I hypothesize that the direct arginylation and acetylation of a-syn fibrils, as shown in aim 1, will reduce the cytotoxicity of a-syn fibrils in Parkinson’s models. I additionally hypothesize that direct phosphorylation of a-syn fibrils, as shown in aim 2, will exacerbate the toxicity of a-syn fibrils in Parkinson’s models. The demonstration of cytoprotective PTMs of a-syn would point to future directions for Parkinson’s therapy; the demonstration of the cytotoxicity of a-syn phosphorylation would clarify the chain of causality between a-syn phosphorylation and cytotoxicity, providing valuable insight into the role of a-syn phosphorylation in Parkinson’s pathogenesis.