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S. M. Noorwez, S. Kaushal; Dose-Dependent Differential Effect of HDAC Inhibitors on Yields of Folded P23H and WT Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5979.
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To determine whether folding of P23H and WT rhodopsin can be increased with the use of histone deacetylase (HDAC) inhibitors (HDIs).
To assess the effect of different HDIs on rhodopsin yields, WT and P23H opsins were expressed separately in tetracycline-inducible stable HEK293 cell lines. The optimum dose of valproic acid (VPA) and trichostatin A (TSA) for increasing opsin yield was determined by administering these inhibitors to the cells in various concentrations. Acetylation of histone 3 was determined by western blots. The optimum dose of each inhibitor was then added to confluent plates of the cells. After 48 hours of incubation, the folded proteins were immunoaffinity-purified and quantitated by UV-visible spectroscopy. The proteins were also examined by immunoblotting. The effect of HDIs on the expression of opsin and other genes was studied by real-time PCR.
The HDIs inhibited HDAC and increased the acetylation of histone 3 in a dose-dependent manner. Total opsin levels for both P23H and WT opsin also increased with increasing HDI concentrations. Overall, HDI treatment led to a two-fold increase in the total opsin levels. However, the increase in the folded protein was between 50-80%, signifying that there was an overall increase in both the folded and the misfolded opsin. At lower concentrations used, both HDIs resulted in the 50-80% increase in the folded rhodopsin. With increasing concentrations, however, the yields of folded P23H rhodopsin were significantly reduced, while the yields of WT rhodopsin increased. Interestingly, the HDIs increased the expression of only the induced rhodopsin from the cells; the expression of the intrinsic opsin gene present in HEK cells was not affected.
Our data demonstrate that HDIs affect the folding of rhodopsin in a dose-dependent manner. At lower concentrations, HDIs increase the yield of folded P23H as well as WT rhodopsin. At higher concentrations, inhibition of HDACs selectively affects the folding of the mutant rhodopsin but not the WT rhodopsin. This differential effect of HDIs may help ameliorate misfolding-related retinitis pigmentosa.
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