Prins, Stella;
(2020)
Can two wrongs make a right? Investigating F508del-CFTR rescue with second-site mutations using a new fluorescence assay for high content CFTR screening.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that is primarily expressed in epithelial cells where it regulates the transepithelial transport of salt and water. Mutations in the CFTR gene can cause cystic fibrosis, and a deletion of the phenylalanine at site 508, F508del, is the most common mutation associated with cystic fibrosis. F508del impairs both biogenesis and gating of CFTR. Instead of being trafficked to the cell membrane, F508del-CFTR is almost completely retained at the endoplasmic reticulum (ER) where it is eventually degraded. The little F508del-CFTR that makes it to the cell membrane has decreased stability and defective gating. This thesis describes the development and validation of a fluorescence-based assay capable of fast and simultaneous quantification of CFTR channel function and membrane proximity in live human embryonic kidney (HEK-293) cells. We constructed a pIRES2-mCherry-YFPCFTR plasmid that directs co-expression of mCherry and CFTR with a halide-sensitive YFP (YFP(H148Q/I152L)) tagged to its N-terminal. The mCherry expression makes it possible to identify the borders of cells, corresponding to the location of the cell membrane. YFP(H148Q/I152L)-CFTR that colocalises with the border, is used to estimate the amount of CFTR in close proximity of the membrane. Function of CFTR is quantified by analysis of the rate of YFP quenching in response to the influx of iodide. We used the assay to systematically search for mutations in cis with F508del that have the potential to rescue F508del-CFTR biogenesis and function. Scanning of a section of intracellular loop 4 (ICL4) with 61 second-site mutations, validates R1070W (Thibodeau et al., 2010) as a particularly effective revertant. Furthermore, we tested the effects of two mutations corresponding to revertant mutations for F670del-Yor1p, a yeast homolog to F508del-CFTR in which the deletion of F670 causes defects in Yor1p similar to those caused by F508del in CFTR.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Can two wrongs make a right? Investigating F508del-CFTR rescue with second-site mutations using a new fluorescence assay for high content CFTR screening |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10115977 |
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