Saturation mutagenesis of α-synuclein reveals monomer fold that modulates aggregation

Author:

Chlebowicz Julita1ORCID,Russ William12ORCID,Chen Dailu1ORCID,Vega Anthony13ORCID,Vernino Steven45ORCID,White Charles L.156ORCID,Rizo Josep789ORCID,Joachimiak Lukasz A.158ORCID,Diamond Marc I.145ORCID

Affiliation:

1. Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, TX, USA.

2. Evozyne Inc., Chicago, IL, USA.

3. Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

4. Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

5. Peter O'Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.

6. Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

7. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

8. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.

9. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Abstract

α-Synuclein (aSyn) aggregation underlies neurodegenerative synucleinopathies. aSyn seeds are proposed to replicate and propagate neuronal pathology like prions. Seeding of aSyn can be recapitulated in cellular systems of aSyn aggregation; however, the mechanism of aSyn seeding and its regulation are not well understood. We developed an mEos-based aSyn seeding assay and performed saturation mutagenesis to identify with single-residue resolution positive and negative regulators of aSyn aggregation. We not only found the core regions that govern aSyn aggregation but also identified mutants outside of the core that enhance aggregation. We identified local structure within the N terminus of aSyn that hinders the fibrillization propensity of its aggregation-prone core. Based on the screen, we designed a minimal aSyn fragment that shows a ~4-fold enhancement in seeding activity and enabled discrimination of synucleinopathies. Our study expands the basic knowledge of aSyn aggregation and advances the design of cellular systems of aSyn aggregation to diagnose synucleinopathies based on protein conformation.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

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