Abstract
Purpose :
Rhodopsin (Rho) is a visual GPCR expressed in rod photoreceptors, which main function is the absorption of a light photon, its transduction into the cell and conversion into the nervous impulse. Mutations in Rho are linked with various ocular impairments. Thus, efforts are directed towards developing novel ligands targeting Rho, improving folding and stability of Rho. However often, small molecule drugs also cause toxicity. Therefore, natural compounds present in plants could be another viable approach in drug discovery efforts. The dietary polyphenol compounds, present in fruits and vegetables have beneficial effects in several eye diseases due to their antioxidant, anti-inflammatory or anti-apoptotic properties. In this study, we aimed to clarify the effect of flavonoids on rod opsin stability, function and membrane organization.
Methods :
We tested four, most common bioactive flavonoids: quercetin, myricetin and their mono-glycosylated forms. Molecular docking was used to predict the binding site of these compounds within the structure of bovine rod opsin. Effects of these flavonoids on pigment regeneration and G protein activation and light-induced chromophore release were tested in a Trp fluorescence assays. The effect on opsin stability was determined in a thermal shift assay using BFC fluorescence probe. The effect of flavonoids on the rod opsin oligomeric organization was investigated in situ in cells expressing rod opsin using BRET, SDS-PAGE, and immunoblotting.
Results :
All four compounds could accommodate into the retinal binding pocket. Additionally, quercetin and myricetin could bind to the external binding pocket. Experimental results indicated that these flavonoids upon binding to opsin affect the rates of isoRho regeneration. Gt activation was slightly enhanced by quercetin and myrcetin at physiological concentrations. However, none of these compounds affected considerably the rates of chromophore release. Both, quercetin and myrcetin, but not their glycosylated forms, increased the stability of opsin. Interestingly, quercetin but not myricetin increased opsin dimerization/oligomerization state within the cell membrane.
Conclusions :
Together, these studies provide evidence that flavonoids can modulate functional and structural properties of rod opsin, and could be beneficial in disease conditions leading to the excessive concentrations of free and misfolded opsin.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.