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

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.

SUBMITTER: Chlebowicz J

PROVIDER: S-EPMC10610913 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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

Chlebowicz Julita J Russ William W Chen Dailu D Vega Anthony A Vernino Steven S White Charles L CL Rizo Josep J Joachimiak Lukasz A LA Diamond Marc I MI

Science advances 20231027 43

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 ...[more]

PMID: 37889966

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Project description:Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH abusers are at high risk of neurodegenerative disorders, including Parkinson's disease (PD). Previous studies have demonstrated that METH causes alpha-synuclein (α-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of α-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of α-syn is involved in high-dose METH-induced α-syn aggregation. We measured the levels of α-syn SUMOylation and these enzymes involved in the SUMOylation cycle in SH-SY5Y human neuroblastoma cells (SH-SY5Y cells), in cultures of C57 BL/6 primary mouse neurons and in brain tissues of mice exposure to METH. We also demonstrated the effect of α-syn SUMOylation on α-syn aggregation after METH exposure by overexpressing the key enzyme of the SUMOylation cycle or silencing SUMO-1 expression in vitro. Then, we make introduced mutations in the major SUMOylation acceptor sites of α-syn by transfecting a lentivirus containing the sequence of WT α-syn or K96/102R α-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT α-syn or K96/102R α-syn into the mouse striatum. Levels of the ubiquitin-proteasome system (UPS)-related makers ubiquitin (Ub) and UbE1, as well as the autophagy-lysosome pathway (ALP)-related markers LC3, P62 and lysosomal associated membrane protein 2A (LAMP2A), were also measured in SH-SY5Y cells transfected with lentivirus and mice injected with adenovirus. The results showed that METH exposure decreases the SUMOylation level of α-syn, although the expression of α-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in α-syn SUMOylation by UBC9 overexpression relieves METH-induced α-syn overexpression and aggregation, whereas the decrease in α-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of α-syn also aggravate α-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of α-syn plays a fundamental part in α-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Dataset Information

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

Publications

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

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