Combinatorial effects of PTMs on a-Synuclein structure function and aggregation

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

α-Synuclein is a small neuronal protein that is the primary component of the proteinaceous aggregates that are the hallmark of Parkinson’s disease and other synucleinopathies. Recent evidence supports the idea that structural differences between α-Synuclein aggregates, or ‘strains’, underlie different synucleinopathies. While the molecular details are not yet well-understood, it has been suggested that post-translational modifications to α-Synuclein may underlie conformational differences between ‘strains’. However, understanding how these modifications impact aggregate structure, and ultimately pathology, is extremely challenging, both given the large number of reported post-translational modifications to α-Synuclein, as well as their heterogeneous distribution in patient-derived samples. From a biochemical and biophysical perspective, many of these modifications have been addressed individually and found to have striking impacts on α-Synuclein properties, including aggregation kinetics and cellular uptake and seeding. However, there is a significant gap in our understanding of how multiple simultaneous modifications may work cooperatively to alter aggregate structure or α-Synuclein function. Our proposed research will address this deficit by taking advantage of the collective expertise of the three PIs in protein chemical synthesis, cellular and molecular biophysics, and structural biology. This will include using a novel semi-synthesis strategy – combining unnatural amino acid mutagenesis, chemoenzymatic modification, thiol-ene reactions, and native chemical ligation – to produce α-Synuclein site specifically modified both at single and multiple sites (Aim 1); determining the impact of α-Synuclein modifications on functional interactions with lipid bilayers, on the kinetics of self-association and on the structural features of the aggregates (Aim 2); and relating these structural effects to internalization of α-Synuclein by primary neurons, and subsequent seeded aggregation of endogenous α-Synuclein (Aim 3). We have selected seven different disease-associated sites on α-Synuclein that are subject to modification with diverse groups, including phosphorylation, acetylation and ubiquitination, and we will compare and contrast the individual effects of these modifications as well as their cross-talk. Through this research we expect to characterize the impact of these modifications both on α-Synuclein functional interactions as well as fibrillar structure and spread. Ultimately, the impact of this work will be in providing a thorough understanding of the molecular basis of ‘strain’ differences in synucleinoapthies and may guide the development of therapies targeted at post-translational modifications, or even open the door to new therapeutic strategies.