New insights into the molecular mechanisms of rhodopsin retinitis pigmentosa
- Autores: Mònica Aguilà Cerdà
- Directores de la Tesis: Pere Garriga Solé (dir. tes.)
- Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2010
- Idioma: inglés
- Tribunal Calificador de la Tesis: Juan Jesús Pérez González (presid.), Núria Saperas Plana (secret.), Esteve Padrós i Morell (voc.), Joan Manyosa Ribatallada (voc.), Michael Cheetham (voc.)
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- Resumen
Rhodopsin is the visual photoreceptor responsible for dim light vision. Mutations in rhodopsin are the most common cause of the severe inherited neurodegenerative blindness, retinitis pigmentosa (RP).
One of the goals of this work was to establish the relationship between the structure and the function of RP mutants, mainly using spectroscopic techniques. Briefly, the study was focused on rhodopsin mutations that affect the ligand bindind pocket. The principal interest was to determine the illumination response and the ability to activate transducin with the rhodopsin mutants. The results showed retinal regeneration problems as well as low activation rates. The next step was to use the retinal analog 11-cis-7 methylretinal to try to unravel the conformational changes that take place when rhodopsin moves to its active form. The results obtained with the combined use of a 7-methyl retinal analog and rhodopsin mutants highlight the tight coupling between the chromophore and the receptor for optimal structure and stability of dark and active conformations of the rod photosensitive pigment. The results with the M207 mutants confirm the specific role of this amino acid in the correctly folded conformation of the receptor and in the activation process.
Another main outline of the project was the study of the cellular trafficking of RP rhodopsin mutants G114D, M207R and R135L. An extensive cellular characterization in cell culture models was carried out. Several pharmacological compounds were tested in this model in an attempt to improve their folding or trafficking defects. Manipulation of molecular chaperones, treatment with chemical chaperones or heat shock protein inducers were used to reduce inclusion incidence, alleviate cell death and promoted the translocation of the mutant protein to the plasma membrane.
To explore different ways of analyzing effects of rhodopsin mutations on its function, we set up the use of Surface Plasmon Resonance as a new tool for studying activation of recombinant rhodopsin. Our success in the immobilization of recombinant rhodopsin allows not only the study of transducin activation but also the possibility to determine the kinetic data of rhodopsin- transducin interaction. The use of this tool can be applied for future studies of recombinant rhodopsin mutants.
