Molecular triggers of disease-related synuclein aggregation in the Parkinson’s environment
Creators: name: Moriarty, Gina M.
Publisher: name: No Publisher Supplied
Year: 2017

description: The works in this thesis explores the challenges involved in recapitulating the environment in which the synuclein family of proteins resides, functions and are active participants in Parkinson’s disease (PD) pathophysiology. The link between α-synuclein (αS or αsyn) aggregation and PD neurodegeneration has been established by its presence in fibrillar form in intracellular inclusions known as Lewy Bodies (LBs) and associated oligomerization, whereas β-synuclein (βS or βsyn) is thought to be a non-fibrillogenic partner to αS, beneficial to disease. The enclosed work covers two main topics surrounding the environmental sensitivities of these intrinsically disordered proteins’ (IDPs) role in generating disease-associated aggregation: 1) the impact of N-terminal acetylation on αS fibrillation, and 2) the pH responsive source of βS’ inhibited nature. First we generate an N-terminally acetylated αS in response to an investigation of the native oligomeric state of αS in the physiological environment. We find that N-terminal acetylation has minimal impact on the disordered monomeric ensemble of αS and that no evidence of a preference for a tetrameric or other kind of oligomer is found.  We find also that acetylated αS forms fibrils of in a similar timeframe and morphology as the non-acetylated form of the protein. While the impact on our view of αS as an intrinsically disordered monomer ensemble is relatively non-consequential, we go on to show that with at least one binding partner, Cu2+, N-terminal acetylation can have a significant impact in vivo. Cu2+ had been shown in vitro to have a unique accelerating effect on αS fibrillation at low, physiological stoichiometries, and given the redox active nature of the metal, and the role of metal dyshomeostasis in neurodegeneration, it had been garnering interest as a significant player in disease-associated αS aggregation. However, we go on to show that N-terminal acetylation occurs at the most significant of three Cu2+ binding sites, blocks binding of the metal and abolishes any fibrillation accelerating effect. Therefore, this work forces reconsideration of how the N-terminally acetylated αS interacts with partners in vivo, and the ultimate role of Cu2+ as an exacerbator of αS pathophysiological aggregation, which may still occur through two other, although much weaker, binding sites at its histidine and C-terminus.  In the second part of this thesis, we discover that the model for the physiological environment typically used to draw the conclusion that βS is non-fibrillogenic in contrast to αS, may not be adequate to use to infer things about its intracellular behavior. We discover that mildly acidic pH 5.8, achievable in several intracellular sub-environments, turns on a fibrillation switch and allows βS to form fibrils. We use this pH-responsiveness and a set of α/βS domain-swapped chimeras to study the roots of βS’ less fibril prone nature. We discover through the chimeras that the NAC domain is the most significant determinant of aggregation behavior, and through elimination of pH sensitive sites, and Rosetta modeling of βS on an αS fibril structure, that acidic side chains can modulate the on/off fibrillation switch. This is further demonstrated in pH insensitive Glu→Ala mutants and the pH-responsiveness is suggested to be rooted in glutamate side chains. This work implies that a more complex role for βS in disease and its partnership with αS may be possible than currently understood, and previously undetected.  These two projects emphasize that when considering IDP misfolding in neurodegeneration in vitro, it is difficult to remove the surface-exposed IDP from its environment in which its behavior is so intricately entwined, but also provides some insight into factors we must consider in our models, including: co/post-translation modifications, binding partners, and solution environment pH.