Ultrasensitive Measurement of Ca 2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

Autor: Flagmeier P; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., De S; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., Wirthensohn DC; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., Lee SF; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., Vincke C; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium., Muyldermans S; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium., Knowles TPJ; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., Gandhi S; Department of Molecular Neuroscience, Institute of Neurology, University College London, Queen Square, London, WC1N 3BG, UK., Dobson CM; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK., Klenerman D; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2017 Jun 26; Vol. 56 (27), pp. 7750-7754. Date of Electronic Publication: 2017 May 05.
DOI: 10.1002/anie.201700966
Abstrakt: To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca 2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca 2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca 2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
(© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Databáze: MEDLINE
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