Structural and functional effects of natural phenolic compounds on rhodopsin mutants associated with retinitis pigmentosa
- Autores: María Guadalupe Herrera Hernández
- Directores de la Tesis: Pere Garriga Solé (dir. tes.)
- Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Juan Jesús Pérez González (presid.), Eulalia Bosch Presegue (secret.), Mònica Aguilà Cerdà (voc.)
- Programa de doctorado: Programa de Doctorado en Polímeros y Biopolímeros por la Universidad Politécnica de Catalunya
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
Dietary polyphenols represent a group of secondary metabolites which widely occur in fruits, vegetables, wine, tea, extra virgin olive oil, chocolate and other cocoa products. Previous studies have shown the interaction of certain phenolic compounds with estrogen and adenosine receptors, belonging to the G-protein coupled receptors (GPCRs) superfamily. GPCRs are the largest family of signal transduction molecules involved in most of the physiological processes.
Rhodopsin is the prototypic member of GPCRs. This receptor is the major protein found in the disks membrane of the outer segments of retinal rod photoreceptor cells and the first with resolved crystal structure. Mutations in rhodopsin are associated with retinitis pigmentosa (RP), a group of inherited visual diseases causing retinal degeneration due to progressive loss of rod and cone photoreceptors cells and leading to blindness. Several studies have been carried out on rhodopsin RP mutants in order to elucidate the molecular mechanism of the disease. This is the first step before suitable therapeutic approaches can be developed. Some of the proposed treatments are based on pharmacological rescue, in which small molecules known as chemical or pharmacological chaperones bind and stabilize misfolded opsins. Polyphenols have been proposed as useful agents against retinal toxicity but no clear evidence of these compounds at the molecular level of the visual phototransduction System has been presented so far.
Given the interest in finding new ligands to compensate the deleterious effects of RP mutations, the aim of this thesis was to evaluate the effect of the polyphenols on the structure and function of the visual pigment rhodopsin RP mutants and to study their preferences to bind to rhodopsin.
Herein we found that polyphenols quercetin (Q), resveratrol (R) and epigallocatechin gallate decreased the expression of wild-type and mutant rhodopsins when added to eukaryotic cell cultures. However, no differences were observed in the biophysical and functional properties of immunopurified pigments regenerated with 11-cis-retinal chromophore treated with O and R al concentrations of 1 µM and 10 µM Our molecular docking complementary analysis indicated that the polyphenol compounds studied can bind to rhodopsin and could act as allosteric ligands for rhodopsin regenerated with 9-cis-retinal as a chromophore.
Our results showed that Q improved the percentage and rate of regeneration of opsin with 9-cis-retinal when compared to 11-cis-retinal. Q enhances the structural compaction around the Schiff Base linkage between the retinal chromophore and opsin, preventing the chemical retinal trapping reagent hydroxylamine from easily entering in the retinal pocket. Moreover, functional experiments on wild-type and G90V mutant rhodopsin regenerated with 9-cis-retinal in presence of 1 µM Q, showed a sigmoidal kinetics clearly representative of cooperative binding. Furthermore, the presence of Q bound to rhodopsin was demonstrated by HPCL-MS analysis.
In summary, it has been shown, by using complementary molecular biology and analytical methods and in silico computational studies that some polyphenol compounds, and particularly quercetin, can act as allosteric modulators of 9-cis-rhodopsin. This effect is particularly significant in the case of the G90V mutation associated with the retinal degenerative disease RP, where the deleterious properties of the mutation could be partially compensated, and opens novel possibility of using such compounds in the treatment of visual neurodegeneration such as that associated to RP.
