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S. M. Noorwez, S. Kaushal; Effect of Small Molecule Inhibitors on Cellular Pathways That Control the Fate of P23H Rhodopsin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1271. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether P23H rhodopsin yields can be increased by selectively modulating cellular pathways that affect P23H opsin.
To assess the effect of perturbation of different cellular pathways on rhodopsin yields WT and P23H opsins were expressed separately in tetracycline-inducible stableHEK293 cell lines. MG132 was added to the cells to perturb proteasomal function. Lysosomal degradation was examined by incubating the cells with ammonium chloride. Anterograde transport from the endoplasmic reticulum was inhibited by increasing concentrations of brefeldin A. The calnexin/calreticulin folding cycle in the ER was disrupted by the addition of castanospermine. Inhibitor concentrations were optimized to maximize the yields and limit cellular toxicity. After 48h of incubation the folded protein were immunoaffinity purified and quantitated by UV-visible spectroscopy.
The small molecules that act at the level of ER e.g. castanospermine and brefeldin A and ammonium chloride displayed comparable effect on both WT and P23H opsins. The increase in the yield of the folded rhodopsins was between 1.3 to 1.5 times. The inhibition of proteasomal pathway, on the other hand, preferentially increased the recovery of the P23H rhodopsin by 2.5 to 3 fold.
Our data demonstrates that modulating folding environment in the ER leads to more P23H rhodopsin. These observations also suggest that different degradation pathways participate to different extents in the processing of P23H opsin. It appears lysosomal degradation contributes to a lesser extent than proteasomal degradation in determining the fate of P23H opsin. By manipulating the protein quality control systems within the cell, it may be possible to alleviate the cellular burden of misfolded P23H opsin.
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