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Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

ABSTRACT: Because oligomers and aggregates of the protein ?-synuclein (?S) are implicated in the initiation and progression of Parkinson's disease, investigation of various ?S aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, ?-sheet-rich fibrillar species by incubation of ?S in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of ?S, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly ?-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type ?S and the C-terminal truncation mutant ?S1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type ?S, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that ?S is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced ?S aggregation both in vitro and in vivo.

SUBMITTER: Anderson VL

PROVIDER: S-EPMC2973859 | biostudies-other | 2010 Nov

REPOSITORIES: biostudies-other

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Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

Anderson Valerie L VL Ramlall Trudy F TF Rospigliosi Carla C CC Webb Watt W WW Eliezer David D

Proceedings of the National Academy of Sciences of the United States of America 20101014 44

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient produc ...[more]

PMID: 20947801

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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 (?-syn) aggregation in the both laboratory animal and human. In this study, exposure to high METH doses increased the expression of ?-syn and the small ubiquitin-related modifier 1 (SUMO-1). Therefore, we hypothesized that SUMOylation of ?-syn is involved in high-dose METH-induced ?-syn aggregation. We measured the levels of ?-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 ?-syn SUMOylation on ?-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 ?-syn by transfecting a lentivirus containing the sequence of WT ?-syn or K96/102R ?-syn into SH-SY5Y cells and injecting an adenovirus containing the sequence of WT ?-syn or K96/102R ?-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 ?-syn, although the expression of ?-syn and SUMO-1 are increased. One possible cause is the reduction of UBC9 level. The increase in ?-syn SUMOylation by UBC9 overexpression relieves METH-induced ?-syn overexpression and aggregation, whereas the decrease in ?-syn SUMOylation by SUMO-1 silencing exacerbates the same pathology. Furthermore, mutations in the major SUMOylation acceptor sites of ?-syn also aggravate ?-syn overexpression and aggregation by impairing degradation through the UPS and the ALP in vitro and in vivo. These results suggest that SUMOylation of ?-syn plays a fundamental part in ?-syn overexpression and aggregation induced by METH and could be a suitable target for the treatment of neurodegenerative diseases.

Dataset Information

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

Publications

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

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