Characterization of the phosphomimetic mutant Y39E of α-Synuclein

α-Synuclein (aSyn) has long been identified as a key factor in the pathogenesis of Parkinson's Disease (PD), with familial mutations in the SNCA gene contributing to the aberrant aggregation of aSyn. Its aggregation results in the formation of Lewy Bodies, a characteristic hallmark of PD, ultimately leading to neurodegeneration and cell loss. While mutations have been extensively studied, post-translational modifications (PTMs), particularly phosphorylation, have emerged as crucial players in aSyn's physiological function and pathological aggregation. The kinase c-abl is known to phosphorylate aSyn predominantly at Tyrosine 39 (Y39), a modification often observed in advanced PD stages associated with elevated c-abl levels. Given the challenges in purifying phosphorylated proteins, phosphomimetic mutants, in this case Y39E, serves to simulate phosphorylation effects. This study extensively characterizes the Y39E aSyn mutant across various aspects, including monomeric structure and dynamics, membrane binding, in vitro and in vivo aggregation properties, dopaminergic neurodegeneration, and toxicity. Employing a multidisciplinary approach, biophysical methods such as nuclear magnet resonance (NMR), circular dichroism (CD) spectroscopy, Thioflavin-T (ThT) fluorescence measurements, and size exclusion chromatography (SEC) were combined with biological methods including molecular cloning, protein expression, and in vivo investigations using cell-based assays and the animal model C. elegans. Structural analysis revealed that the Y39E monomer closely resembled the wild-type (WT) monomeric structure, with no significant differences in backbone dynamics nor in the hydrodynamic radius. However, slightly higher R2 relaxation rates and minor changes in transient long-range interactions were observed in the Y39E variant. The overall membrane affinity remained equal between WT and Y39E, although the NAC region of Y39E exhibited reduced interaction. Notably, during the aggregation process, Y39E incorporated fewer monomers and displayed a prolonged lag phase with concentration-dependent kinetics. In cellular and animal models, the Y39E mutant demonstrated fewer cellular inclusions and smaller aggregates, respectively. Compared to the WT species, dopaminergic neurodegeneration was significantly elevated when expressing Y39E aSyn in C. elegans, impacting the nematode's behavior, while mitochondrial pathways were ruled out as a cause of toxicity. Additionally, data acquired on pY39 aSyn by other research groups aligned closely with results obtained in this study, indicating the Y39E mutant as a valuable tool for probing phosphorylation at this specific site, and confirming that pY39 can be mimicked by the Y39E mutant. Furthermore, the significance of pY39 is underscored by evidence of various research groups, showing that inhibiting phosphorylation effectively hinders disease progression and diminishes pathology in animal models. In summary, this work characterizes de Y39E mutant of aSyn. The structure and dynamics of the Y39E aSyn monomer closely resembled those of the WT species. However, differences were observed in the aggregation profiles between WT and Y39E aSyn, and dopaminergic neurodegeneration was found to be elevated for the Y39E mutant. Furthermore, this work highlights the crucial role of PTMs in aSyn pathology, showing that mutations, such as Y39E emerge as valuable instruments for mimicking PTMs, thereby simplifying the research process.