Allosteric modulation of mutant rhodopsins folding and function as a new therapeutic strategy for the treatment of retinitis pigmentosa
(English) G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in mammals and are essential for sensing external signals and regulating intracellular signaling pathways. Among GPCRs, rhodopsin plays a critical role in phototransduction in retinal rod cells, and its structu...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/695400 |
| Acceso en línea: | http://hdl.handle.net/10803/695400 https://dx.doi.org/10.5821/dissertation-2117-443014 |
| Access Level: | acceso abierto |
| Palabra clave: | G protein-coupled receptors rhodopsin trace metals retigabine allosteric modulation retinitis pigmentosa Àrees temàtiques de la UPC::Enginyeria química Àrees temàtiques de la UPC::Ciències de la visió 577 - Bioquímica. Biologia molecular. Biofísica 617 - Cirurgia. Ortopèdia. Oftalmologia |
| Sumario: | (English) G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in mammals and are essential for sensing external signals and regulating intracellular signaling pathways. Among GPCRs, rhodopsin plays a critical role in phototransduction in retinal rod cells, and its structural and functional integrity is vital for vision. Mutations in the opsin gene are closely associated with inherited retinal diseases such as retinitis pigmentosa (RP), characterized by progressive photoreceptor degeneration and eventual blindness. Despite limited therapeutic options for RP, emerging strategies focusing on protein stabilization and allosteric modulation offer new hope. This study systematically explored the effects of trace metal ions and small molecules on rhodopsin stability, chromophore regeneration dynamics, and signaling properties. We found that Fe³⁺ significantly enhances the thermal and chemical stability of both wild-type (WT) and G90D mutant, while Fe²⁺ exhibited no protective effects and Cu²⁺ strongly destabilized rhodopsin and impaired its regeneration. These findings highlight the critical role of metal ion homeostasis in maintaining rhodopsin function and suggest that modulating iron oxidation states could be a therapeutic strategy for retinal degenerative diseases. Analysis of RP-associated G90D and G90V mutants revealed that while both mutations caused a blue shift in the absorption maximum, G90V exhibited markedly reduced thermal stability, impaired photobleaching response, delayed retinal release, and severely disrupted chromophore regeneration, whereas G90D showed less altered structural and functional characteristics. Although Fe³⁺ provided some stabilization to G90D, it was less effective than in WT rhodopsin, likely due to mutation-induced conformational changes. Retigabine significantly enhanced the thermal stability of rhodopsin and promoted efficient chromophore regeneration, indicating its potential as a pharmacological chaperone for stabilizing rhodopsin mutants implicated in retinal degenerative diseases. Retigabine bound rhodopsin without disrupting photobleaching kinetics or G-protein activation, likely binding to an allosteric site to stabilize protein conformation while preserving normal phototransduction. These properties highlight retigabine potential as a therapeutic strategy for retinal degeneration. Moreover, the study analyzed the F88L mutation, revealing that F88L rhodopsin maintains normal spectral properties and visual function but exhibits enhanced thermal stability and accelerated MII decay. Hydroxylamine unexpectedly stabilized the MI intermediate in F88L, indicating a novel regulatory aspect of rhodopsin photocycle. These results suggest that amino acid at position 88 may be involved in rhodopsin molecular evolution and in light adaptation in nocturnal vision. In addition, the small molecule C24 was found to modulate F88L rhodopsin by enhancing chromophore regeneration and accelerating MII decay, indicating potential strategies for fine-tuning rhodopsin dynamics. Overall, these findings provide important insights into the stabilization mechanisms of rhodopsin and lay a foundation for developing novel therapies targeting retinal degenerative diseases through metal ion regulation and small-molecule modulation. |
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