Kseniia Afitska1, Anna Fucikova2, Volodymyr V Shvadchak3, Dmytro A Yushchenko4. 1. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, Prague 16610, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Albertov 6, Prague 12843, Czech Republic. 2. Department of Chemical Physics & Optics, Faculty of Mathematics & Physics, Charles University, Ke Karlovu 3, Prague 12116, Czech Republic. 3. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, Prague 16610, Czech Republic. 4. Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo namesti 2, Prague 16610, Czech Republic; Miltenyi Biotec GmbH, Friedrich-Ebert-Straße 68, Bergisch Gladbach 51429, Germany. Electronic address: yushchenko@uochb.cas.cz.
Abstract
BACKGROUND: Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with the protein concentration. For the development of efficient inhibitors of α-synuclein aggregation, it is important to know the critical concentration of aggregation (the concentration of monomeric protein, below which the protein does not aggregate). METHODS: We performed in vitro aggregation studies of α-synuclein at low concentrations (0.11-20 μM). Aggregation kinetics was measured by ThT fluorescence. Obtained aggregates were characterized using CD-spectroscopy, fluorescent spectroscopy, dynamic light scattering and AFM imaging. RESULTS: Monomeric α-synuclein at concentrations 0.45 μM and above was able to bind to fibril ends resulting in fibril growth. At the protein concentrations below 0.4 μM, monomers did not fibrillize, and fibrils disaggregated. In the absence of seeds, fibrils were formed only at monomer concentrations higher than 10 μM. At low micromolar concentrations, we observed formation of prefibrillar amyloid aggregates, which are able to induce fibril formation in α-synuclein solutions of high concentrations. CONCLUSIONS: The critical concentration of α-synuclein fibril growth is ~0.4 μM. Prefibrillar amyloid aggregates appear at concentrations between 0.45 and 3 μM and are an intermediate state between monomers and fibrils. Although morphologically different from fibrils, prefibrillar aggregates have similar properties to those of fibrils. GENERAL SIGNIFICANCE: We determined the critical concentration of α-synuclein fibril growth. We showed that fibrils can grow at much lower monomer concentrations than that required for de novo fibril formation. We characterized a prefibrillar intermediate species formed upon aggregation of α-synuclein at low micromolar concentration.
BACKGROUND: Aggregation of the neuronal protein α-synuclein into amyloid fibrils is a hallmark of Parkinson's disease. The propensity of α-synuclein to aggregate increases with the protein concentration. For the development of efficient inhibitors of α-synuclein aggregation, it is important to know the critical concentration of aggregation (the concentration of monomeric protein, below which the protein does not aggregate). METHODS: We performed in vitro aggregation studies of α-synuclein at low concentrations (0.11-20 μM). Aggregation kinetics was measured by ThT fluorescence. Obtained aggregates were characterized using CD-spectroscopy, fluorescent spectroscopy, dynamic light scattering and AFM imaging. RESULTS: Monomeric α-synuclein at concentrations 0.45 μM and above was able to bind to fibril ends resulting in fibril growth. At the protein concentrations below 0.4 μM, monomers did not fibrillize, and fibrils disaggregated. In the absence of seeds, fibrils were formed only at monomer concentrations higher than 10 μM. At low micromolar concentrations, we observed formation of prefibrillar amyloid aggregates, which are able to induce fibril formation in α-synuclein solutions of high concentrations. CONCLUSIONS: The critical concentration of α-synuclein fibril growth is ~0.4 μM. Prefibrillar amyloid aggregates appear at concentrations between 0.45 and 3 μM and are an intermediate state between monomers and fibrils. Although morphologically different from fibrils, prefibrillar aggregates have similar properties to those of fibrils. GENERAL SIGNIFICANCE: We determined the critical concentration of α-synuclein fibril growth. We showed that fibrils can grow at much lower monomer concentrations than that required for de novo fibril formation. We characterized a prefibrillar intermediate species formed upon aggregation of α-synuclein at low micromolar concentration.
Authors: Anne-Laure Mahul-Mellier; Johannes Burtscher; Niran Maharjan; Laura Weerens; Marie Croisier; Fabien Kuttler; Marion Leleu; Graham W Knott; Hilal A Lashuel Journal: Proc Natl Acad Sci U S A Date: 2020-02-19 Impact factor: 11.205
Authors: Enrico Zurlo; Pravin Kumar; Georg Meisl; Alexander J Dear; Dipro Mondal; Mireille M A E Claessens; Tuomas P J Knowles; Martina Huber Journal: PLoS One Date: 2021-01-22 Impact factor: 3.240