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R. Sama, S. M. Noorwez, S. Kaushal; Role of Heat Shock Response in Rhodopsin Folding. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5980.
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To determine the effect of the heat shock response (HSR) on the folding of P23H and WT rhodopsin.
To assess the effect of different heat shock inducers (HSIs) on rhodopsin yields, WT and P23H opsins were expressed separately in tetracycline-inducible stable HEK293 cell lines. The optimum dose of two HSIs, celastrol and geldanamycin, for increasing opsin yield was determined by administering these inducers to the cells in various concentrations. The cellular levels of Hsp70 were determined by western blots. The optimum dose of each inducer was then added to confluent plates of the cells. After 48 hours of incubation, the folded protein was immunoaffinity-purified and quantitated by UV-visible spectroscopy.
The HSIs increased the levels of Hsp70 in the cells. The presence of celastrol increased Hsp70 levels by 20%; geldanamycin increased Hsp70 levels by 60%. Total opsin levels for both P23H and WT opsin increased with increasing HSI concentrations. Addition of geldanamycin increased WT opsin levels by 15-20% while P23H opsin levels increased by 45-50%. When assessed for its effect on folded rhodopsin, addition of geldanamycin increased folded WT rhodopsin levels by 10%. In sharp contrast, P23H rhodopsin levels increased by 40% in the presence of geldanamycin. Addition of celastrol increased total opsin levels to 10% and 40% for WT and P23H, respectively. Celastrol increased the levels of folded WT rhodopsin by only 5%. In the case of P23H, celastrol increased folded rhodopsin levels by 35% above the placebo controls.
Our data demonstrate that induction of the HSR by small molecules increases the yields of folded P23H rhodopsin. The effect of HSIs appears to be more selective for mutant P23H than for WT rhodopsin. This increase of folded rhodopsin may help ameliorate misfolding-related retinitis pigmentosa.
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