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Modification of C Terminus Provides New Insights into the Mechanism of alpha-Synuclein Aggregation
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SYSNO ASEP 0485440 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Modification of C Terminus Provides New Insights into the Mechanism of alpha-Synuclein Aggregation Author(s) Afitska, Kseniia (UOCHB-X) ORCID, RID
Fučíková, A. (CZ)
Shvadchak, Volodymyr V. (UOCHB-X) ORCID, RID
Yushchenko, Dmytro A. (UOCHB-X) ORCID, RIDSource Title Biophysical Journal. - : Cell Press - ISSN 0006-3495
Roč. 113, č. 10 (2017), s. 2182-2191Number of pages 10 s. Language eng - English Country US - United States Keywords alpha-synuclein ; aggregation ; kinetics Subject RIV BO - Biophysics OECD category Biophysics Institutional support UOCHB-X - RVO:61388963 UT WOS 000416213100010 EID SCOPUS 85029605070 DOI 10.1016/j.bpj.2017.08.027 Annotation 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). Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Viktorie Chládková, Tel.: 232 002 434 Year of Publishing 2018
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