Investigating the effects of predicted stabilizing secondary mutations in nucleotide binding domain 2 of CFTR-N1303K
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Joseph Dakota Taylor (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Robert Youker
Abstract: Cystic Fibrosis is a disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. This membrane protein selectively transports chloride ions into epithelial cells and is important in a wide array of excretory functions. Mutations that hinder CFTR biogenesis, transport, or gating are the underlying cause of Cystic Fibrosis (CF). Impairment of CFTR function can lead to numerous health problems such as lung fluid buildup, pancreatitis, and bowel blockages. The most common mutation is a deletion of F508 (?F508) that resides in NBD1 of CFTR but rarer mutations in NBD2 can also lead to disease. For example, N1303K is a rarer mutation in Caucasian populations, but is more common in Mediterranean and Middle Eastern populations. Recent computational experiments from the Youker lab have identified potential stabilizing mutations in the mutant N1303K. The second site mutations L1346F and L1254A were analyzed for this thesis. The MutPred2 analyses of N1303K-CFTR predicted that L1346F mutation would greatly reduce pathogenicity, while L1254A would only have a modest effect. FoldX corroborates this as well, with a ??G value of 2.3 kcal/mol upon mutation to L1254A and -1.26 kcal/mol change upon mutation of L1346F. The results of molecular dynamic (MD) simulations using NBD2 domain revealed different results with the L1254A having a closer RMSF, RMSD, hydrogen bonds and solvent accessible surface area similar to wildtype CFTR, while L1346F had characteristics closer to N1303K, suggesting that L1346F might not be beneficial. Principle component analysis corroborates MD results, with L1254A having a similar protein movement pattern to wildtype CFTR. Site-directed mutagenesis was used to introduce L1254A and L1346F mutations into a plasmid containing GFP-Tagged N1303K-CFTR. The mutated plasmids were transiently transfected into HEK-293 cells and in-gel fluorescence performed on cellular lysates. The L1346F mutant was slightly stabilized as determined by C/B band ratio. However, a higher amount of L1254A mutant protein was present in the plasma membrane compared to L1346F and N1303K-CFTR as determined by image colocalization analysis. The L1346F mutant had more internal staining and this could indicate aggregated protein, but further cellular and biochemical experiments are needed to confirm. Overall, these results suggest the L1254A mutant has enhanced trafficking to the cell surface compared to N1303K or L1346F, but there is enhanced proteolysis of this double mutant as seen in protein gels. It is not clear if the proteolysis occurs in the cell, or post-lysis. Further cellular experiments with lysosomal and/or proteasomal inhibitors may shed light on when proteolysis occurs.
Investigating the effects of predicted stabilizing secondary mutations in nucleotide binding domain 2 of CFTR-N1303K
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Created on 6/1/2023
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2023
- Subjects
- Nucleotides
- Cystic fibrosis
- Mutation (Biology)
- Molecular dynamics
- Biochemistry