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Anthony Michael Chiu, Jake Mandziuk, Joseph R Casey; Identifying Chemical Correctors for the Folding Defect in Corneal Dystrophy-Causing Mutants of SLC4a11. Invest. Ophthalmol. Vis. Sci. 2014;55(13):496.
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Congenital Hereditary Endothelial Dystrophy (CHED) and Fuchs Endothelial Corneal Dystrophy (FECD) are two forms of genetic corneal blindness. CHED is recessive whereas FECD is dominant with a 4% occurrence. Some cases of these diseases are caused by mutations in the membrane protein, SLC4a11, which has been identified as forming a water translocation pathway. SLC4a11 at the plasma membrane facilitates water flow out of the stroma and into the aqueous humor. Mutant SLC4a11 is non-functional because it is either retained in the endoplasmic reticulum or is catalytically dead. Current treatments for these diseases, including corneal transplant, are wholly inadequate. Our goal is to identify compounds that could be used as drugs to correct these protein folding defects.
We have developed a simple, high-throughput assay to identify chemical correctors that enable diseased SLC4a11 to target to the plasma membrane. SLC4a11 with a hemagglutinin (HA) epitope tag inserted into an extracellular loop was stably expressed in HEK293 cells, seeded on a 96 well plate and then incubated with the potential correcting compound of choice. Cells were then incubated with anti-HA antibody, followed by secondary antibody conjugated to horseradish peroxidase (HRP). In the presence of H2O2, HRP metabolizes AmplexRed to create a red fluorescence product, read in a 96 well plate reader. A higher relative amount of fluorescence indicates a higher proportion of protein at the plasma membrane. Mutants being tested include G709E (FECD), A269V, E143K and R869C (CHED).
The ability of the assay to differentiate plasma membrane-rescued versus ER-retained SLC4a11 was assessed by measurement of fluorescence arising from cells expressing WT protein with an intracellular or extracellular HA tag. Fluorescence was six-fold higher in cells expressing the extracellular tagged protein. Compounds screened for their ability to correct SLC4a11 misfolding include, DMSO, glycerol, 4-phenylbutyric acid and the Hsp90 inhibitor, geldanamycin.
We have developed an assay that enables rapid, sensitive and accurate screening of compounds that correct the folding defect in SLC4a11 mutants. Further screening of potential correcting compounds already approved for use in humans, holds potential to identify pharmaceutical based treatment for FECD and CHED.
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