Modification of C Terminus Provides New Insights into the Mechanism of alpha-Synuclein Aggregation

0485440 - ÚOCHB 2018 RIV US eng J - Journal Article
Afitska, Kseniia - Fučíková, A. - Shvadchak, Volodymyr V. - Yushchenko, Dmytro A.
Modification of C Terminus Provides New Insights into the Mechanism of alpha-Synuclein Aggregation.
Biophysical Journal. Roč. 113, č. 10 (2017), s. 2182-2191. ISSN 0006-3495. E-ISSN 1542-0086
Institutional support: RVO:61388963
Keywords : alpha-synuclein * aggregation * kinetics
OECD category: Biophysics
Impact factor: 3.495, year: 2017

Aggregation of neuronal protein alpha-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of alpha-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to alpha-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such alpha-synuclein sequence extension on alpha-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of alpha-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of alpha-synuclein. To address these questions, we prepared mutants of alpha-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type alpha-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of alpha-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of alpha-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).
Permanent Link: http://hdl.handle.net/11104/0280467