von Kolontaj, Kerstin: Fast detection of protein-protein interactions with an automated FRET-based system on the flow cytometer. - Bonn, 2018. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-50116
@phdthesis{handle:20.500.11811/7522,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-50116,
author = {{Kerstin von Kolontaj}},
title = {Fast detection of protein-protein interactions with an automated FRET-based system on the flow cytometer},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2018,
month = mar,

note = {Although protein-protein interactions play a major role in almost all biological functions, those interactions cannot easily be analysed, especially not on single cell level and with regard to dynamic changes in protein arrangement. Conventional investigation methods, like the yeast two-hybrid system are highly time consuming. Förster resonance energy transfer (FRET) is commonly used to identify protein-protein interactions via confocal microscopy. However, this requires expert knowledge and produces high amounts of data.
In order to overcome those limitations, a program was developed that automatically measures and calculates the FRET efficiency on cell by cell basis on the MACSQuant flow cytometer. Changes in protein-protein interactions can be assessed via relative signal changes of the donor and acceptor fluorochromes, causing a change in FRET efficiency. This allows for the identification of protein-protein interactions on large cell numbers in a minimum of time, in high throughput screenings and is easy to use.
In this study, it could shown that interaction of the CD3 and CD4 coreceptors can be measured on the T cell’s surface after activation via an increase in FRET efficiency. Furthermore, also the homoclustering of the CD3 coreceptor could be detected using the automatic FRET measurement. Even when the FRET fluorochrome pair was changed, the results were highly comparable. For this experiment, the increase in FRET efficiency was even faster than the increase in intracellular calcium that is used as the standard activation marker. For that reason, this automatic assay had been used to test blood samples of patients that are suffering from severe immunodeficiencies and are compromised for different reasons in their ability of CD3 clustering. Indeed, here no increase in FRET efficiency could be measured compared to healthy blood donors. Therefore, the automatic FRET program can easily be used in clinical settings for the determination of certain immunodeficiencies in patients.
Moreover, using the CD3 homoclustering FRET essay, the impact on lipid raft integrity on the clustering of CD3 could be detected. If the lipid rafts were manipulated, the CD3 receptor could not cluster effectively after T cell stimulation, leading to a decreased FRET efficiency compared to untreated T cells.
The automatic FRET assay could also be used to determine the checkpoint inhibition in T cells. For that purpose, the interaction of CD3 and the PD-1 receptor was measured after a prolonged T cell stimulation leading to an increased FRET efficiency. That increase could be inhibited by applying blocking antibodies that are directed against the PD-1 ligands, prohibiting the co-stimulation that is essential for a successful immune escape. Therefore, this method is also a powerful tool allowing for high throughput screenings for pharmacologically active compounds.},

url = {http://hdl.handle.net/20.500.11811/7522}
}

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