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Liudmila Cebotaru, Miqueias Lopes-Pacheco, Inna Sabirzhanova, Daniele Rapino, Rahul Grover, James T Handa, William Guggino; Rescuing ABCA4 trafficking mutants as a treatment for Stargardt disease.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5465.
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© ARVO (1962-2015); The Authors (2016-present)
Stargardt macular degeneration, which invariably ends in legal blindness and has no known cure, is caused by mutations in abca4, a member of the ABC family. Cystic fibrosis is caused by mutations in CFTR, a related ABC protein. We tested the hypothesis, using cells stably expressing the disease causing mutants, R1108C and R1129C, that correctors which rescue the homologous cystic fibrosis causing CFTR mutation, ΔF508-CFTR, can be used to treat certain abca4 mutations.
Stable HEK293 cell lines were created using Flp-In (Life Technologies). Small-molecule correctors, C18, CFFT and VX-809 (Selleckchem.) were applied (1-20 μM) for 16h. Western blots and coimmunoprecipitation were performed using ABCA4, HDAC6 and Hsp27 antibodies. Cell surface expression was assessed by biotinylation and translation inhibited by cycloheximide.
R1108C and R1129C express 60% less protein than wt ABCA4. Large increases in mutant protein levels, compared to wt, occurred in cells grown at 27oC, demonstrating that both mutants are temperature sensitive. When translation was inhibited, mutant proteins rapidly disappeared whereas, wt-ABCA4 remained stable. Temperature-sensitivity and rapid disappearance indicate that the mutant proteins are unstable and rapidly degraded. Correctors VX-809 or C18, designed to rescue the most common CFTR mutant, ΔF508, increased total protein and cell surface expression of R1108C and R1129C by 1.5 and 2 fold, respectively. Studies with cellular quality control proteins indicated that there is an approximately 1.5 and 5 fold greater binding of HDAC6 (histone deacetylase) and Hsp27 (heat shock protein), respectively, to R1108C and R1129C compared to wt. When treated with C18 or VX-809, HDAC6 and Hsp27 binding to R1108C and R1129C was significantly reduced. Thus, correctors VX-809 and C18 rescue total and surface protein expression of R1108C and R1129C, and reduce their binding to HDAC6 and Hsp27, two proteins involved in their degradation.
Correctors identified to rescue ΔF508-CFTR will rescue ABCA4 mutants selected within regions of greatest similarity between the two ABC proteins. Because VX-809 is already in clinical trial for cystic fibrosis, our results provide a potential pathway for treating Stargardt disease.
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