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Updated: May 27, 2025
A Method to Study α-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model
Published on: November 25, 2022
Structurally targeted mutagenesis identifies key residues supporting α-synuclein misfolding in multiple system
Patricia M Reis1,2, Sara Am Holec1,3, Chimere Ezeiruaku1,4
1Department of Biology and Institute for Applied Life Sciences, University of Massachusetts Amherst, Amherst, MA, USA.
Background:
Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α-synuclein spreading throughout the central nervous system. While familial PD is linked to several α-synuclein mutations, no mutations are associated with MSA. We previously showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that α-synuclein adopts unique strains in patients.
Objective:
Here we sought to further interrogate α-synuclein misfolding to identify the structural determinants that contribute to MSA strain biology.
Methods:
We engineered a panel of cell lines harbouring both PD-linked and novel mutations designed to identify key residues that facilitate α-synuclein misfolding in MSA. We also used Maestro in silico analyses to predict the effect of each mutation on α-synuclein misfolding into one of the reported MSA cryo-electron microscopy conformations.
Results:
In many cases, our modelling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α-synuclein misfolding.
Conclusions:
Our studies used a structure-based approach to investigate α-synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.
