Manipulating the intracellular fate of an aggregate-prone protein





Shalesh Kaushal

University of Florida, Department of Ophthalmology, 1600 Archer Road, Gainesville, Florida 32610-0284, USA



Like many inherited and acquired protein conformational diseases (PCDs), P23H opsin associated with Autosomal Dominant Retinitis Pigmentosa (ADRP) is largely a misfolded protein and retained within the cell. We had previously described a set of pharmacological chaperones, including the native chromophore 11-cis-retinal, that quantitatively promoted the in vivo folding and stabilization of P23H opsin. Like the wild-type (WT) protein, the rescued mutant formed pigment, acquired mature glycosylation, and was transported to the cell surface. We now demonstrate that by inhibiting calnexin activity by the inhibitor castanospermine or inhibiting endoplasmic reticulum (ER) to Golgi transport by Brefeldin A lead to 1.3-1.5 times greater cellular yields of the folded mutant protein. However, kifusineine, a compound known to promote ER- associated degradation reduced the yield of folded P23H. Additionally, the inhibitors of the proteasome or autophagy led to even greater yields, increasing the folded form by 2.5-3.0 times, while their effect on the WT protein was minimal. To our knowledge this is the first demonstration that by titrating the inhibition of various cellular processes, including the quality control systems of the cells, the amount of a mutant folded protein can be substantially increased to wild-type levels. In conjunction with pharmacological chaperones, such an approach may serve as a useful adjuvant therapy for PCDs.




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