Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis : phosphorylation at Tyrosine 125 and monoubiquitination at Lysine 6

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the firs