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Title: Restoring structure and function in rhodopsin autosomal dominant retinitis pigmentosa mutants
Author: Opefi, Chikwado Alo
Awarding Body: University of Essex
Current Institution: University of Essex
Date of Award: 2011
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Autosomal dominant retinitis pigmentosa is a genetic disorder that leads to progressive vision loss and blindness. Over 150 AD RP mutations are located in rhodopsin; a gene that encodes the dim light photoreceptor pigment of the rod cell. Previous studies indicate that a large proportion of rhodopsin AD RP mutants fail to fold correctly. Here, we have constructed all known ADRP mutations in the N- , terminus of rhodopsin in a synthetic bovine gene, and have also made other ADRP mutations located at the transmembrane domain. We have examined their expression and folding in HEK-293S cells and have attempted to restore the misfolding phenotype by different approaches. We found that pharmacological rescue with 11-cis retinal promotes folding in all AD RP mutants in the N-terminus, but to different extents. However, the rescued purified pigments remain thermo-unstable and are defective in photobleaching and signal transduction. j As an alternative approach, this set of mutants was put into rhodopsin mutant N2C/D282C: a background which is more thermostable than WT rhodopsin due to formation of a disulfide bond between the two introduced cysteines. We found that some, but not all, AD RP mutants are repaired in this background and form a visible, pigment. In contrast to the 11 -cis retinal rescued pigments, the 2C-282C repaired pigments have enhanced stability and have near normal photobleaching and signal transduction properties. Our major conclusion from this work is that the N-terminal cap of rhodopsin performs a key role in the folding and stabilization of rhodopsin as well as in its signal transduction activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available