Chemical Chaperone Modulation of Mutant Opsin Folding and Processing

Abstract

Abstract: : Purpose: Mutations in rhodopsin cause autosomal dominant retinitis pigmentosa (RP). In vitro studies of transfected cells in culture have previously shown that wild–type (WT) rod opsin translocates to the plasma membrane whereas the P23H mutation misfolds, is retained within the endoplasmatic reticulum (ER), is degraded by the ubiquitin proteasome system (UPS), and forms intracellular inclusions. This study was carried out in order to test the hypothesis that chemical chaperones can assist in the folding and processing of P23H mutant rod opsin. Methods: SH–N–SH neuroblastoma cells were transiently transfected with GFP–tagged WT and P23H rod opsin (WT–opsin–GFP and P23H–opsin–GFP). The transfected cells were treated with a variety of chemical chaperones at a range of concentrations. Fluorescence microscopy was used to determine the incidence of opsin inclusions. The processing of opsin within the cell was also analysed by confocal microscopy and biochemically. Results: Chemical chaperones were found to reduce inclusion formation in P23H–opsin–GFP opsin expressing cells. Furthermore, some chemical chaperones led to an increase the amount of mutant protein that was processed to the plasma membrane. Conclusions: The data suggest that chemical chaperones can assist P23H rod opsin folding and/or degradation resulting in reduced inclusion formation and consequent mutant protein translocation to the plasma membrane. Therefore, chemical chaperones may be of use in facilitating the production of mutant opsins for cellular and biochemical analyses of their pathogenic consequences and could also be of use therapeutically to improve mutant rod opsin folding in rhodopsin RP patients.

Keywords: opsins • retinal degenerations: cell biology • chaperones