Quantifying the Proteolytic Cleavage of Plasma Membrane Proteins in Living Cells
Autori: Calamai M., Pavone F.S.
Affiliazione autori: National Institute of Optics, National Research Council of Italy (CNR), Florence, Italy; LENS – European Laboratory for Non-linear Spectroscopy, Florence, Italy
Abstract: The standard approach to study the activity of proteases consists of lysing cells and measuring the changes in the fluorescence properties of a synthetic substrate after cleavage in vitro. Here, a general protocol that uses a bi-fluorescent chimeric construct of a known substrate protein that follows the proteolytic processing in living cells is described. This approach is useful, in particular, to search for pharmacological conditions altering the cleavage rate of a certain protease, or to investigate the biological factors influencing a certain proteolytic mechanism. Three different methods (microscopy, flow cytometry, and spectroscopy) to detect fluorescence changes due to alteration in the processing are described. This approach was originally developed for studying conditions affecting the proteolytic activity of the ß-secretase Bace1 on the amyloid precursor protein APP, but can in principle be applied to investigate any membrane protein undergoing ectodomain shedding by proteolytic cleavage. © 2018 by John Wiley & Sons, Inc.
Volume: 81 (1) Da Pagina: e58-1 A: e58-11
Maggiori informazioni: The research leading to these results has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 654148 Laserlab-Europe (EU-H2O20 654148), from the Italian Ministry of Health in the frame-work of the project “Automated digital scanning and diagnosis of tissues using multimodal non-linear optical microscopy” (GR-2011-02349626), from the Italian Ministry for Education, University and Research in the frame-work of the Flagship Project NANOMAX.Parole chiavi: amyloid precursor protein; beta secretase 1, amino terminal sequence; Article; cell membrane; clinical protocol; confocal microscopy; controlled study; flow cytometry; high throughput screening; human; human cell; microscopy; priority journal; protein analysis; protein cleavage; protein degradation; protein domain; protein processing; SH-SY5Y cell line; spectrofluorometry; spectroscopy; surgical technique