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postgraduate thesis: Regional disturbances by the [delta]F508 mutation on the cystic fibrosis transmembrane conductance regulator

TitleRegional disturbances by the [delta]F508 mutation on the cystic fibrosis transmembrane conductance regulator
Authors
Issue Date2016
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Chen, X. [陳新穎]. (2016). Regional disturbances by the [delta]F508 mutation on the cystic fibrosis transmembrane conductance regulator. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractCystic fibrosis (CF) is a genetic disease caused by dysfunction of the cystic fibrosis transmemberane conductance regulator (CFTR). The ΔF508 mutation, missing the phenylalanine residue at position 508, is the most common mutation carried by about 90% patients with CF. The ΔF508 mutation causes two major defects in CFTR, including impaired protein trafficking from endoplasmic reticulum to Golgi apparatus and reduced channel activity. However, it is unclear whether ΔF508-induced CFTR defects may be associated with disturbance on function of neighbor residues. To test this hypothesis, we measured protein expression of serial CFTR deletion mutants, each with a residue deleted from position 491 to 525. Immunoblotting data demonstrate that most deletion mutants like ΔF508-CFTR exhibited immature protein with only core glycosylation from ER shown as Band B, whereas ΔV510- and ΔS511-CFTR presented abundant expression of mature protein with complex glycosylation from Golgi apparatus shown as Band C, which runs to a position higher than Band B in electrophoresis gel. To understand whether the processing defects of these deletion mutations are caused by lack of the backbone or the side chain in a residue, we tested several mutants each with a single alanine-substitution from position 503 to 515. Our data demonstrate that most mutants exhibited abundant Band C expression, whereas N505A- and D513A-CFTR showed little and moderate Band C expression, respectively. Moreover, CFTR Band C%, intensity ratio of Band C versus (Band C + Band B), was greatly reduced in all mutants with residue substitutions at the N505 or R560 residues, and also in the mutant M498P-CFTR, but not M498A- nor M498F-CFTR. These data suggest that the side-chain interactions of the N505 residue with R560’s side chain or M498’s backbone are important for normal CFTR processing. To test whether some deletion mutations caused the processing defects by the mechanism similar to that in ΔF508-CFTR, we treated these mutants with low-temperature culture and CFTR correctors, which can enhance Band C expression of ΔF508-CFTR. Our data demonstrate that low-temperature culture and corrector C18 enhanced Band C% of ΔF508-, ΔV510-, ΔS511- and ΔY512-CFTR. In addition, combined treatments together with correctors C4, C18 and low-temperature culture showed additive effects on promoting Band C expression of ΔF508- and ΔY512-CFTR than the treatment with a single effector. These data suggest that the ΔF508 and ΔY512 mutations may impair CFTR processing by a similar mechanism. Using site-directed mutations in the loop between the F508 and Y512 residue, we found that the side chains of the G509 and V510 residues may contribute to the mechanism that causes ΔF508-CFTR processing defect. Finally, our study on the single-channel activity of wild-type, ΔF508-, ΔV510-, ΔS511- and ΔY512-CFTR indicates that only ΔF508- and ΔY512-CFTR had reduced open probability and prolonged interburst interval, compared to that of wild-type CFTR. These data demonstrate that ΔF508- and ΔY512-CFTR exhibited similar defects in CFTR channel gating. Taken together, our data suggest that dysfunction of the residues around the F508 residue may partly account for both protein processing and channel gating defects in ΔF508-CFTR.
DegreeDoctor of Philosophy
SubjectCystic fibrosis - Pathophysiology
Dept/ProgramBiomedical Sciences
Persistent Identifierhttp://hdl.handle.net/10722/238831
HKU Library Item IDb5824317

 

DC FieldValueLanguage
dc.contributor.authorChen, Xinying-
dc.contributor.author陳新穎-
dc.date.accessioned2017-02-20T02:06:36Z-
dc.date.available2017-02-20T02:06:36Z-
dc.date.issued2016-
dc.identifier.citationChen, X. [陳新穎]. (2016). Regional disturbances by the [delta]F508 mutation on the cystic fibrosis transmembrane conductance regulator. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/238831-
dc.description.abstractCystic fibrosis (CF) is a genetic disease caused by dysfunction of the cystic fibrosis transmemberane conductance regulator (CFTR). The ΔF508 mutation, missing the phenylalanine residue at position 508, is the most common mutation carried by about 90% patients with CF. The ΔF508 mutation causes two major defects in CFTR, including impaired protein trafficking from endoplasmic reticulum to Golgi apparatus and reduced channel activity. However, it is unclear whether ΔF508-induced CFTR defects may be associated with disturbance on function of neighbor residues. To test this hypothesis, we measured protein expression of serial CFTR deletion mutants, each with a residue deleted from position 491 to 525. Immunoblotting data demonstrate that most deletion mutants like ΔF508-CFTR exhibited immature protein with only core glycosylation from ER shown as Band B, whereas ΔV510- and ΔS511-CFTR presented abundant expression of mature protein with complex glycosylation from Golgi apparatus shown as Band C, which runs to a position higher than Band B in electrophoresis gel. To understand whether the processing defects of these deletion mutations are caused by lack of the backbone or the side chain in a residue, we tested several mutants each with a single alanine-substitution from position 503 to 515. Our data demonstrate that most mutants exhibited abundant Band C expression, whereas N505A- and D513A-CFTR showed little and moderate Band C expression, respectively. Moreover, CFTR Band C%, intensity ratio of Band C versus (Band C + Band B), was greatly reduced in all mutants with residue substitutions at the N505 or R560 residues, and also in the mutant M498P-CFTR, but not M498A- nor M498F-CFTR. These data suggest that the side-chain interactions of the N505 residue with R560’s side chain or M498’s backbone are important for normal CFTR processing. To test whether some deletion mutations caused the processing defects by the mechanism similar to that in ΔF508-CFTR, we treated these mutants with low-temperature culture and CFTR correctors, which can enhance Band C expression of ΔF508-CFTR. Our data demonstrate that low-temperature culture and corrector C18 enhanced Band C% of ΔF508-, ΔV510-, ΔS511- and ΔY512-CFTR. In addition, combined treatments together with correctors C4, C18 and low-temperature culture showed additive effects on promoting Band C expression of ΔF508- and ΔY512-CFTR than the treatment with a single effector. These data suggest that the ΔF508 and ΔY512 mutations may impair CFTR processing by a similar mechanism. Using site-directed mutations in the loop between the F508 and Y512 residue, we found that the side chains of the G509 and V510 residues may contribute to the mechanism that causes ΔF508-CFTR processing defect. Finally, our study on the single-channel activity of wild-type, ΔF508-, ΔV510-, ΔS511- and ΔY512-CFTR indicates that only ΔF508- and ΔY512-CFTR had reduced open probability and prolonged interburst interval, compared to that of wild-type CFTR. These data demonstrate that ΔF508- and ΔY512-CFTR exhibited similar defects in CFTR channel gating. Taken together, our data suggest that dysfunction of the residues around the F508 residue may partly account for both protein processing and channel gating defects in ΔF508-CFTR.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshCystic fibrosis - Pathophysiology-
dc.titleRegional disturbances by the [delta]F508 mutation on the cystic fibrosis transmembrane conductance regulator-
dc.typePG_Thesis-
dc.identifier.hkulb5824317-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiomedical Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.mmsid991021207499703414-

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