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Carolina Uggenti, Lisa Swanton, Forbes D Manson; Use of small molecules to correct mutant bestrophin-1 localization.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6015.
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Bestrophin-1 is a transmembrane protein of the basolateral plasma membrane of the RPE. Human mutations in BEST1 cause bestrophinopathies, characterized by retinal dystrophy and an altered electro-oculogram. Mechanisms through which mutations in bestrophin-1 cause disease remain unknown. Exogenous expression of mutant bestrophin-1 results in reduced chloride channel conductance, mislocalization and protein degradation. Our aims are to characterize the quality control mechanisms (QC) leading to mislocalization and degradation of mutant bestrophin-1 and to identify small molecules that correct localization and function.
Expression of wildtype (WT) and mutant bestrophin-1 was induced in stable MDCKII cell lines with tetracycline. Protein expression and localization was determined by immunoblotting and immunofluorescence respectively. Functional analysis was performed by whole-cell patch-clamping and calcium imaging of FURA2-loaded cells. Cells were treated with lysosomal protease and proteasome inhibitors to identify the QC involved in mutant bestrophin-1 recognition. The potential therapeutic role of small molecules to stabilize and promote normal trafficking and function of mutant bestrophin-1 proteins was investigated by adding them to the culture medium for 24h prior analysis.
Very low levels of mutant bestrophin-1 were detected by immunoblotting compared to WT. However strong immuno-reactive bands for both mutant and WT bestrophin-1 were detected when cells were treated with the proteasome inhibitor PSII or bortezomib (BTZ) suggesting that mutant bestrophin-1 proteins are less stable than the WT and are rapidly degraded via proteasomal degradation pathways. Increased expression of the endogenous chaperone Hsp70 was detected following BTZ treatment, suggesting that BTZ may stabilize bestrophin-1 by inhibiting protein degradation and promote folding by up-regulating Hsp70. Of the other small molecules tested, 4-PBA caused a modest increase in the amount of mutant bestrophin-1 protein. Immunofluorescence showed that a combination of BTZ and small molecules were able to restore trafficking of at least one mutant bestrophin-1 protein to the plasma membrane.
We have demonstrated a rescue of protein expression and trafficking of mutant bestrophin-1 proteins by treatment with combination of small molecules. Functional analyses are on-going.
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