Misfolded rhodopsin mutants display variable aggregation properties

Abstract

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental.

Flavonoids enhance rod opsin stability, folding, and self-association by directly binding to ligand-free opsin and modulating its conformation

“…Interestingly, no effects on oligomer formation were observed in the cells regenerated with 9-cis-retinal prior to the treatment with flavonoids, thus supporting other results indicating that flavonoids bind to and modulate the properties of the ligand-free opsin only. Moreover, the flavonoid-stimulated increased BRET signal occurred due to the formation of opsin oligomers rather than aggregates as they were disrupted with the DDM detergent, in opposition to the RP-linked opsin aggregates that are not sensitive to such treatment (59). Thus, this finding suggests that increased opsin stability could be related to the formation of tightly packed oligomers within the biological membranes.…”

“…Cells were resuspended and transferred from the 96-well cell culture plate to a white-walled opaque 96-well plate (Corning Life Sciences). DDM in PBS at a final concentration of 5 or 0.1 mM was added to the wells (59). Coelenterazine h was diluted to 25 M in PBS from a 2.5 mM stock solution.…”

“…Cells were resuspended, and 200 l of this suspension was transferred to a white-walled opaque 96-well plate (Corning, NY). DDM in PBS at a final concentration of 5 mM or 0.1 mM was added to the assigned wells (59). The measurement of the BRET signal was performed as described above for the stable cell line.…”

Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

“…In Drosophila, RHO mutants can recruit WT RHO into intracellular aggregates (62)(63)(64). In contrast to that, recent studies suggest that in mammalian cells, misfolded RHO P23H does not aggregate with properly folded RHO WT (65,66), although other evidence does suggest there is a dominant-negative effect of RHO P23H on the WT protein (2, 67).…”

The Retinoid and Non-Retinoid Ligands of the Rod Visual G Protein-Coupled Receptor

“…Thus, these in vitro studies suggested the pharmacological potential of cis-retinoids. However as discovered recently, 9-cis-retinal added to the cells co-expressing adRP mutants together with WT opsin resulted in the aggregation of the mutant receptor, indicating that treatment with this pharmacological chaperone could be beneficial for the homozygous patient but rather ineffective or detrimental for heterozygous patients [46]. Therefore, screening for safer, non-retinoid small molecules that would stabilize rod opsin and improve its folding is of urgent importance.…”