Qiangni Liu 1 , Inna Sabirzhanova 1 , Emily Anne Smith Bergbower 1 , Murali Yanda 1 , William G Guggino 1 , Liudmila Cebotaru 2 Show Affiliations »
Abstract
BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease. © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration , which invariably ends in legal blindness . We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A . Both mutants cause a reduction in heat shock protein, Hsp27 . Incubation of HEK293 cells expressing the mutants with VX-809 , an FDA approved drug for the treatment of cystic fibrosis , increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27 . Our results provide a pathway for the treatment of Stargardt disease . © Copyright by the Author(s). Published by Cell Physiol Biochem Press.
Entities: CellLine
Chemical
Disease
Gene
Mutation
Species
Keywords:
Chaperones; Common mutations; Correctors; Rescue; Stargardt disease
Mesh： See more »
Substances： See more »
Year: 2019
PMID： 31403270 PMCID： PMC7027368 DOI： 10.33594/000000146
Source DB: PubMed Journal: Cell Physiol Biochem ISSN： 1015-8987