Characterization of amino acid changes in visual pigment evolution and interaction with associated proteins
- Autores: Miguel Antonio Fernández Sampedro
- Directores de la Tesis: Pere Garriga Solé (dir. tes.)
- Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Juan Jesús Pérez González (presid.), Arnau Cordomi Montoya (secret.), Néstor Gómez Trias (voc.)
- Programa de doctorado: Programa de Doctorado en Tecnología Agroalimentaria y Biotecnología por la Universidad Politécnica de Catalunya
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
Visual opsins are G protein-coupled receptors that function as light photoreceptors in the vertebrate retina. Rhodopsin is the visual pigment located in the rod photoreceptor cells specialized in scotopic vision.
Bovine and mouse rhodopsins have been thoroughly used as in vitro and in vivo models for physiological and biochemical characterization. In the last years, different lines of evidence point to significant functional differences among rhodopsins of different species. In this thesis bovine, murine and human rhodopsins were immunopurified and biochemically characterized, revealing differences in their thermal stabilities and retinal release rates. Besides, the Y102H RP-like rhodopsin mutation was introduced in the human and bovine backgrounds to bring up potential phenotypic differences. Therefore, keeping in mind that a large body of studies on human genetic retinal degenerative diseases related with opsins (e.g. retinitis pigmentosa) have used these models, our results suggest that using human rhodopsin for future studies would be advised.
The most important biochemical differences were observed between the diurnal (human and bovine) versus nocturnal (mouse) species, especially in their retinal release rates. In addition, we also found a novel relevant amino acid position that appears to be significantly correlated with rhodopsin molecular adaptation to the nocturnal (L290) and the diurnal (I290) niches throughout terrestrial therian mammals. Previous studies suggested that L290 is present in the inferred therian ancestor rhodopsin in agreement with mammalian “nocturnal bottleneck” theories. Thus, the L290I substitution could have an important role in mammal rhodopsin molecular evolution and adaptation as it is likely to be the result of independent analogous changes, a fact that can be well-appreciated in the primate and rodent orders. This hypothesis was experimentally confirmed by the L290I mutation in murine rhodopsin that resulted in a Meta II decay rate similar to that of bovine rhodopsin.
These results provide support for a role of the Meta II decay rate in rhodopsin evolution, beyond the well-studied ¿max spectral shift used by animal species to adapt to different light environments. Moreover, a novel mechanism is proposed involving a compromise between improving rod protection under bright light in nocturnal species by means of a stabilized Meta II conformation, and a faster dark adaption that occurs under dim-light conditions in diurnal species by means of a faster retinal reléase.
Our statistical analysis found three new candidate positions for positive selection in the mammal therian branch. The reverse mutations (F13M, Q225R and A346S) were introduced into bovine rhodopsin and the expressed proteins were immunopurified to functionally and biochemically characterize the consequences of these ancestral changes. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Position 13 was shown to be very important for the proper folding and glycosylation of rhodopsin as only in the engineered thermally stable double Cys mutant (N2C/N282C) background was able to be regenerated with 11-cis-retinal. Similarly a double Cys mutation (W90C/A169C) previously proposed for the green cone opsin was biochemically analyzed confirming the formation (at least partially) of this bond.
Finally, a recently detected interaction between the membrane protein peripherin-2 and rhodopsin was functionally studied, showing reduced G-protein activation, by rhodopsin, in presence of peripherin-2 when the two proteins were in a partially solubilised system. These results could have physiological implications in the desensitization process involving rhodopsin on the rim of discs of photoreceptor cells.
