Role of the Lipid Environment and Cholesterol Content in the Function of CFTR Modulators

Cystic Fibrosis (CF), an inherited disorder leading to severe damage to the lungs, is caused by mutations in a gene that codes for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Despite CFTR being a protein located in epithelial cell membranes, it has not been established whether the membrane surrounding CFTR plays a role in the function of the recently-discovered CFTR modulators. In concert with a variety of biophysical and biochemical techniques, this thesis explores the link(s) among the environment, small-molecule drugs, and protein sequence in membrane protein folding and functioning. More specifically, a possible significant role for cholesterol - which makes up 30% of the plasma membrane - in facilitating such drug-CFTR interactions, is considered in detail. As studied in three inter-related projects, we focused on the stabilization/folding of a series of mutant constructs - termed helical hairpins - that consist of two adjacent transmembrane segments of CFTR and the intervening extracellular loop. First, an evaluation of the connection between the environment/solvent and sequence changes was undertaken using mutant and WT CFTR TM3/4 hairpins in the presence of four common membrane mimetics: SDS, LDAO, POPC, and POPC + varying cholesterol concentrations. Then, we evaluated the effects of two CFTR modulators - Ivacaftor and Lumacaftor (and their combination, Orkambi) - on the folding of mutant CFTR hairpins, with a focus on whether these drugs can rescue the fold of the hairpins to the WT conformation. Finally, to gauge the importance of the membrane environment in the function of small-molecule drugs, we explored the link between hairpin-modulator interaction and membrane composition. The results show that the interaction of CFTR modulators with the membrane is highly increased in the presence of cholesterol, while circular dichroism spectroscopy and tryptophan fluorescence quenching experiments suggest that cholesterol promotes the helical fold of the constructs in the membrane. Furthermore, the potentiator Ivacaftor’s interaction with the membrane is highly enhanced in the presence of even small amounts of cholesterol. Liposome disruption assays and differential scanning calorimetry experiments suggest that both Ivacaftor and Lumacaftor penetrate/diffuse through the membranes with high cholesterol concentrations (such as 12.5% and 30% cholesterol, as seen in Golgi apparatus and plasma membrane, respectively). However, they behave differently at low cholesterol concentrations: Ivacaftor still penetrates and fully diffuses through membranes containing 5% cholesterol (representing the endoplasmic reticulum), while Lumacaftor resides in such membranes. When cholesterol is not present in the membrane (0% cholesterol), Ivacaftor does not interact with it, while Lumacaftor tends to reside in the membrane. Our experiments reveal that cholesterol plays an important role in the functioning of CFTR modulators and in the interaction of these drugs with the membrane. Additionally, our findings help to better understand the role of cholesterol in CFTR-modulator interactions, further bridging the gap in knowledge to existing CF drugs and paving the way for the design of more effective CF treatments.

Department dc:contributor.department

Biochemistry

Year dc:date.issued

2024

Author dc:creator

• Ravamehr-Lake, Dorna

Advisor dc:contributor.advisor

• Deber, Charles M

Handle dc:identifier.uri

http://hdl.handle.net/1807/140024

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/140024