eprintid: 10115977 rev_number: 25 eprint_status: archive userid: 608 dir: disk0/10/11/59/77 datestamp: 2020-12-11 12:43:57 lastmod: 2021-12-02 23:05:58 status_changed: 2020-12-11 12:43:57 type: thesis metadata_visibility: show creators_name: Prins, Stella 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 ispublished: inpress divisions: UCL divisions: B02 divisions: C08 divisions: D09 note: 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. 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. date: 2020-11-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_open thesis_award: Ph.D language: eng thesis_view: UCL_Thesis primo: open primo_central: open_green verified: verified_manual elements_id: 1830937 lyricists_name: Prins, Stella lyricists_id: SPRIN45 actors_name: Prins, Stella actors_name: Allington-Smith, Dominic actors_id: SPRIN45 actors_id: DAALL44 actors_role: owner actors_role: impersonator full_text_status: public pagerange: 1-182 pages: 182 event_title: UCL institution: UCL (University College London) department: Neuroscience, Physiology and Pharmacology thesis_type: Doctoral editors_name: Vergani, P citation: 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). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10115977/1/doctoral%20thesis%20Stella%20Prins%20-%20Can%20two%20wrongs%20make%20a%20right.pdf