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Resum Introducció: Les opsines són proteïnes integrals de membrana que presenten absorció selectiva per a cada tipus de fotopigment cobrint tot el rang de la visió humana. Una molècula de fotopigment està formada per una opsina més un cromòfor, el derivat de la vitamina A, retinal-11-cis. Les opsines es localitzen en la segona capa de la retina que conté les cèl·lules fotoreceptores cons i bastonets. En aquestes cèl·lules es on es produeix l’absorció de llum que produeix la reacció fotoquímica en el fotopigment que últimament es transmet en forma d’impuls nerviós al cervell. La rodopsina es la proteïna fotoreceptora responsable de la visió escotòpica a baixes intensitats lumíniques. Recentment s’ha vist que la rodopsina pot jugar un paper en el mecanisme molecular d’altres sentits o processos fisiològics. Objectius: L'objectiu del treball és fer una revisió bibliogràfica de les publicacions el més actual possible sobre el tema i una anàlisi crítica de la versemblança dels resultats presentats en aquestes publicacions. Metodologia: S'ha dut a terme una cerca bibliogràfica entre els mesos de febrer 2023 i desembre de 2023 a través de Web of Science. Per fer aquesta cerca hem utilitzat les següents paraules clau ''Opsin”, “Functions”, “Senses”. Resultats: S’han pogut trobar un nombre limitat d’articles que posen de manifest un possible paper primigeni de les opsines en l’evolució molecular d’altres sentits. Aquesta evolució culminaria en el sofisticat mecanisme de la fototransducció visual. En aquest sentit s’ha descrit la relació de les opsines amb d’altres sentits com ara el sentit del gust o el seu possible paper com a sensors de temperatura. Discussió: Els treballs consultats permeten establir que existiria relació entre altres sentits, diferents de la visió, i les opsines. Es tracta però de pocs treballs i que majoritàriament s’han fet amb organismes model com ara la mosca Drosophila melanogaster. Es posa de manifest que calen més estudis., Resumen Introducción: Las opsinas son proteínas integrales de membrana que presentan absorción selectiva para cada tipo de fotopigmento, cubriendo todo el rango de la visión humana. Una molécula de fotopigmento está formada por una opsina más un cromóforo, el derivado de la vitamina A, retinal-11-cis. Las opsinas se localizan en la segunda capa de la retina que contiene las células fotorreceptoras conos y palillos. En estas células es donde se produce la absorción de luz que produce la reacción fotoquímica en el fotopigmento que últimamente se transmite en forma de impulso nervioso en el cerebro. La rodopsina es la proteína fotorreceptora responsable de la visión escotópica a bajas intensidades lumínicas. Recientemente se ha visto que la rodopsina puede jugar un papel en el mecanismo molecular de otros sentidos o procesos fisiológicos. Objetivos: El objetivo del trabajo es realizar una revisión bibliográfica de las publicaciones lo más actual posible sobre el tema y un análisis crítico de la verosimilitud de los resultados presentados en estas publicaciones. Metodología: Se ha realizado una búsqueda bibliográfica entre los meses de febrero 2023 y diciembre de 2023 a través de Web of Science. Para realizar esta búsqueda hemos utilizado las siguientes palabras clave ''Opsin”, “Functions”, “Senses”. Resultados: Se ha podido encontrar un número limitado de artículos que ponen de manifiesto un posible papel primigenio de las opsinas en la evolución molecular de otros sentidos. Esta evolución culminaría en el sofisticado mecanismo de la fototransducción visual. En este sentido, se ha descrito la relación de las opsinas con otros sentidos como el sentido del gusto o su posible papel como sensores de temperatura. Discusión: Los trabajos consultados permiten establecer que existiría relación entre otros sentidos, distintos de la visión, y las opsinas. Se trata de pocos trabajos y que mayoritariamente se han hecho con organismos modelo como la mosca Drosophila melanogaster., Summary Introduction: Opsins are integral membrane proteins that present selective absorption for each type of photopigment, covering the entire range of human vision. A photopigment molecule is made up of an opsin plus a chromophore, the vitamin A derivative, 11-cis-retinal. Opsins are located in the second layer of the retina that contains the cone and stick photoreceptor cells. It is in these cells that the absorption of light occurs, producing the photochemical reaction in the photopigment that is ultimately transmitted in the form of a nervous impulse in the brain. Rhodopsin is the photoreceptor protein responsible for scotopic vision at low light intensities. Recently it has been seen that rhodopsin may play a role in the molecular mechanism of other senses or physiological processes. Objectives: The objective of the work is to carry out a bibliographic review of the most current publications on the subject and a critical analysis of the plausibility of the results presented in these publications. Methodology: A bibliographic search was carried out between the months of February 2023 and December 2023 through Web of Science. To carry out this search we have used the following keywords ''Opsin”, “Functions”, “Senses”. Results: A limited number of articles have been found that reveal a possible primary role of opsins in the molecular evolution of other senses. This evolution would culminate in the sophisticated mechanism of visual phototransduction. In this sense, the relationship of opsins with other senses such as the sense of taste or their possible role as temperature sensors has been described. Discussion: The works consulted allow us to establish that there would be a relationship between other senses, other than vision, and opsins. These are few works and have mostly been done with model organisms such as the fly Drosophila melanogaster. It is evident that more studies are needed to confirm this role of opsins in the evolution of senses other than vision.

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease., This research was funded by grants from Ministry of Science and Innovation, Spain (PID2019-104817GB-I00), and from the Government of Catalonia to Research Consolidated Groups (2021 SGR 00342) to P.G. F.W. is the recipient of a predoctoral grant from the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia and the European Social Fund (2021 FI_B 00228)., Peer Reviewed, Postprint (published version)

Mutations in the photoreceptor protein rhodopsin are known as one of the leading causes of retinal degeneration in humans. Two rhodopsin mutations, Y102H and I307N, obtained in chemically mutagenized mice, are currently the subject of increased interest as relevant models for studying the process of retinal degeneration in humans. Here, we report on the biochemical and functional characterization of the structural and functional alterations of these two rhodopsin mutants and we compare them with the G90V mutant previously analyzed, as a basis for a better understanding of in vivo studies. This mechanis- tic knowledge is fundamental to use it for developing novel therapeutic approaches for the treatment of inherited retinal degeneration in retinitis pigmentosa. We find that Y102H and I307N mutations affect the inactive–active equilibrium of the receptor. In this regard, the mutations reduce the stability of the inactive conformation but increase the stability of the active conformation. Furthermore, the initial rate of the functional activation of transducin, by the I307N mutant is reduced, but its kinetic profile shows an unusual increase with time suggesting a profound effect on the signal transduction process. This latter effect can be associated with a change in the flexibility of helix 7 and an indirect effect of the mutation on helix 8 and the C-terminal tail of rhodopsin, whose potential role in the functional activation of the receptor has been usually underes- timated. In the case of the Y102H mutant, the observed changes can be associated with conformational alterations affecting the folding of the rhodopsin intradiscal domain, and its presumed involvement in the retinal binding process by the receptor., This research was supported by Grant PID2019-104817GB-I00 from Ministerio de Ciencia e Innovación (MICINN), Peer Reviewed, Postprint (published version)

Aquest treball final de Màster, és una proposta on es pretén donar cobertura a un petit buit que hi ha dins els programes de formació i inserció (PFI). No hi ha cap PFI d'auxiliar en mecanitzat, per tant l'autor plantejarà tres mòduls d'aprenentatge en mecanitzat. Dins d'un curs sencer PFI hi ha tres blocs de formació. Els blocs de formació general i tutoria, tant el temari com les hores, ja venen determinats pel currículum establert per la Generalitat a la RESOLUCIÓ ENS/2250/2014, de 6 d'octubre, de l'estructura dels programes de formació i inserció. Però en canvi, les hores destinades al bloc de formació professional, poden ser entre 360h a 410h. L'autor vol fer la proposta de creació de tres mòduls professionals (MP) sencers i bàsics, i les corresponents Unitats Formatives (UF) necessàries i bàsiques, per tal d'assegurar una formació adequada. L'estructura serà la següent: del bloc destinat a la formació professional es plantegen tres MP, un destinat a la introducció de treballs amb eines manuals i trepants (llima, serra, broques, mascles de roscar, ...). Aquest mòdul es documenta amb més detall i servirà d'exemple per els altres dos. Un altre MP destinat a l'aprenentatge de mecanitzat en màquines convencionals (torn i fresadora) i un darrer MP a la introducció de mecanitzat en màquines CNC. En aquest TFM l'autor també a fet un breu recull d'història i del perquè es van crear els PFI, dels seus antecedents, estructures i el seu àmbit legislatiu.

Rhodopsin is the G protein-coupled receptor of rod photoreceptor cells that mediates vertebrate vision at low light intensities. Mutations in rhodopsin cause inherited retinal degenerative diseases such as retinitis pigmentosa. Several therapeutic strategies have attempted to address and counteract the deleterious effect of rhodopsin mutations on the conformation and function of this photoreceptor protein, but none has been successful in efficiently preventing retinal degeneration in humans. These approaches include, among others, the use of small molecules, known as pharmacological chaperones, that bind to the receptor stabilizing its proper folded conformation. Valproic acid, in its sodium valproate form, has been used as an anticonvulsant in epileptic patients and in the treatment of several psychiatric disorders. More recently, this compound has been tested as a potential therapeutic agent for the treatment of retinal degeneration associated with retinitis pigmentosa caused by rhodopsin mutations. We now report on the effect of sodium valproate on the conformational stability of heterologously expressed wild-type rhodopsin and a rhodopsin mutant, I307N, which has been shown to be an appropriate model for studying retinal degeneration in mice. We found no sign of enhanced stability for the dark inactive conformation of the I307N mutant. Furthermore, the photoactivated conformation of the mutant appears to be destabilized by sodium valproate as indicated by a faster decay of its active conformation. Therefore, our results support a destabilizing effect of sodium valproate on rhodopsin I307N mutant associated with retinal degeneration. These findings, at the molecular level, agree with recent clinical studies reporting negative effects of sodium valproate on the visual function of retinitis pigmentosa patients., This research was supported by grant PID2019-104817GB-I00 from Ministerio de Ciencia e Innovación (MICINN)., Peer Reviewed, Postprint (published version)

Dietary polyphenols are a group of natural compounds that have been proposed to have beneficial effects on human health. They were first known for their antioxidant properties, but several studies over the years have shown that these compounds can exert protective effects against chronic diseases. Nonetheless, the mechanisms underlying these potential benefits are still uncertain and contradictory effects have been reported. In this review, we analyze the potential effects of polyphenol compounds on some visual diseases, with a special focus on retinal degenerative diseases. Current effective therapies for the treatment of such retinal diseases are lacking and new strategies need to be developed. For this reason, there is currently a renewed interest in finding novel ligands (or known ligands with previously unexpected features) that could bind to retinal photoreceptors and modulate their molecular properties. Some polyphenols, especially flavonoids (e.g., quercetin and tannic acid), could attenuate light-induced receptor damage and promote visual health benefits. Recent evidence suggests that certain flavonoids could help stabilize the correctly folded conformation of the visual photoreceptor protein rhodopsin and offset the deleterious effect of retinitis pigmentosa mutations. In this regard, certain polyphenols, like the flavonoids mentioned before, have been shown to improve the stability, expression, regeneration and folding of rhodopsin mutants in experimental in vitro studies. Moreover, these compounds appear to improve the integration of the receptor into the cell membrane while acting against oxidative stress at the same time. We anticipate that polyphenol compounds can be used to target visual photoreceptor proteins, such as rhodopsin, in a way that has only been recently proposed and that these can be used in novel approaches for the treatment of retinal degenerative diseases like retinitis pigmentosa; however, studies in this field are limited and furthe, Peer Reviewed, Postprint (published version)

Les patologies corneals són una de les principals causes de ceguesa al món. El tractament tradicional és un trasplantament de còrnia amb una còrnia de donant humà, i en casos més específics, la implantació d’una còrnia artificial. Aquests tractaments però, tenen limitacions importants: per una banda, hi ha una gran escassetat de donants corneals, el que genera llargues llistes d’espera, i per altra banda, tant en el trasplantament corneal com en la implantació de còrnies artificials hi ha un alt risc de complicacions i rebuig. Per aquest motiu, hi ha la necessitat de buscar una solució efectiva i cada vegada més grups d’investigació busquen aquesta solució a través de l’enginyeria tissular i la regeneració de teixits. En aquest treball, es fa una recerca bibliogràfica per identificar les principals limitacions de les opcions actuals de tractament, així com les opcions de regeneració corneal a través dels biomaterials més importants i amb més perspectives de futur. Aquestes estratègies de regeneració corneal són molt diverses i utilitzen biomaterials naturals, sintètics, o un barreges d’ambdós. En qualsevol cas, tot i els recents avenços en aquest camp, no s’ha trobat encara la tècnica ideal, però es preveu trobar-la en un futur proper, i segurament sigui una combinació de dues o més de les estratègies analitzades

La Quercetina (Q) es un flavonoide presente en frutas y vegetales, así como en el té, café, cacaoy vino tinto. La ingesta de Qen la dieta se ha relacionado con efectos beneficiosos en la salud, podemos nombrar entre una multitud,la protección frente a enfermedades cardiovasculares, menor incidencia en la aparición de determinados tipos de cáncer, menor incidencia de asma y de enfermedad pulmonar obstructiva crónica. Las propiedades beneficiosas de la Q se relacionan estrechamente con su estructura química, que le confiere propiedades antioxidantes. Actúa como protector frente a las especies reactivas de oxígeno mediante la neutralización de radicales libres. El efecto antioxidante también podría deberse a su capacidad para inhibir enzimas como la xantina oxidasa, lipooxigenasa y NADPH oxidasa, impidiendo la muerte celular. Además puede incrementar la producción de antioxidantes endógenos (Vicente-Vicente et al 2013).En este trabajo analizaremos y discutiremos algunos estudios que se han llevado a cabo sobre la administración de la Qen la prevención de la salud visual.Stefek (2011) revisa el papel de los flavonoides en la prevención de las cataratas diabéticas. Kalt W. et al (2010) discuten el papel de los compuestos polifenólicos en la salud del ojo. Recientes estudios in vitrodemuestran que las antocianinas y otros flavonoides interactúandirectamente con la rodopsina y modulan la función deestepigmento visual. Otros estudios muestran que los flavonoides protegen una variedad de célulasretinianas de la muerte celular inducida por estrés oxidativo.La Q disminuye la inflamación en cultivos de tejido orbital entero, se cree que actúa como un antioxidante queprotege el epitelio pigmentario de la retina (RPE) dañadopor el estrés oxidativo. Se ha sugerido que los polifenoles vegetales son fármacos prometedores para tratar el cáncer y las enfermedades de la retina

La melanopsina és una proteïna d'un tipus específic de cèl·lules ganglionars de la retina que és activada per la llum i té un paper rellevant en el rellotge biològic que regula els ritmes circadians i el son.Treball bibliogràfic de 6 crèdits.

La retinosis pigmentaria (RP) es un grupo clínico y genéticamente heterogéneo de trastornos de la retina hereditarios caracterizados por una disfunción progresiva difusa de fotorreceptores predominantemente bastón, con la posterior degeneración de fotorreceptores de cono y epitelio pigmentario retiniano. La discapacidad visual generalmente se manifiesta como ceguera nocturna y pérdida progresiva del campo visual. Su prevalencia es de 1:3000 a 1:5000. La RP puede verse de forma aislada (RP típica) o en asociación con enfermedad sistémica. El presente trabajo se enfoca en la RP típica con una breve mención de tipos de RP raros pero con posibles opciones terapéuticas y las diferentes formas de abordarlas. Existe una variación considerable en la gravedad clínica de estas afecciones; El tratamiento actual de la RP incluye un intento de desacelerar el proceso degenerativo a través de terapias. Las terapias novedosas incluyen el uso de terapia génica a medida, trasplante de células madre o tratamiento de neuroprotección. Se han explorado muchos tratamientos sin beneficio comprobado para las formas aisladas de RP. Estos incluyen varias vitaminas y minerales, vasodilatadores, terapia de tejidos con extracto placentario, cortisona, simpatectomía cervical, inyecciones de un hidrolizado, factor de transferencia, dimetilsulfóxido, ozono, trasplantes musculares e inyecciones subretinianas de células retinianas fetales. A pesar de estos avances, no existe un tratamiento efectivo disponible en la actualidad Tras una revisión de la literatura actual exhaustiva se ha podido concluir que los recientes avances en la terapia génica y la tecnología de células madre han posicionado el campo al borde de lo que muchos creen que será un gran avance en la estrategia terapéutica. La tecnología de prótesis retiniana ha permitido restaurar la vista a los ojos completamente ciegos "sin percepción de luz", una hazaña que hace poco tiempo se veía como imposible. La terapia génica basada en líneas d

Vertebrate vision starts with light absorption by visual pigments in rod and cone photoreceptor cells of the retina. Rhodopsin, in rod cells, responds to dim light, whereas three types of cone opsins (red, green, and blue) function under bright light and mediate color vision. Cone opsins regenerate with retinal much faster than rhodopsin, but the molecular mechanism of regeneration is still unclear. Recent advances in the area pinpoint transient intermediate opsin conformations, and a possible secondary retinal-binding site, as determinant factors for regeneration. In this Review, we compile previous and recent findings to discuss possible mechanisms of ligand entry in cone opsins, involving a secondary binding site, which may have relevant functional and evolutionary implications, Peer Reviewed, Postprint (author's final draft)

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 rat, Los opsinas visuales son receptores acoplados a proteína G que funcionan como fotoreceptores en retinas de vertebrados. La rodopsina es el pigmento visual de los bastones, células fotoreceptoras especializadas en la visión escotópica. Las rodopsinas bovina y murina han sido ampliamente usadas como modelos para caracterización bioquímica y fisiológica. En esta tesis, las rodopsinas bovina, murina y humana fueron inmunopurificadas y caracterizadas bioquímicamente, revelando diferencias en su estabilidad térmica y en la tasa de salida de retinal. Además, la mutación tipo RP Y102H se introdujo en las rodopsinas humana y bovina para revelar potenciales diferencias fenotípicas. Teniendo en cuenta que una gran parte de estudios en enfermedades genéticas degenerativas de la retina humana relacionadas con opsinas (Ej. Retinitis Pigmentosa) han usado estos modelos, los resultados sugieren que el uso de rodopsinas humanas en estudios futuros sería aconsejable. Las mayores diferencias bioquímicas fueron observadas entre especies diurnas (humano y vaca) en comparación con la nocturna (ratón), especialmente en las tasas de salida de retinal. Además, se encontró una nueva y relevante posición aminoacídica que parece estar significativamente correlacionada con la adaptación molecular de la rodopsina a la nocturnidad (L290) y a la diurnidad (I290) a lo largo de los mamíferos terios terrestres. Estudios previos sugieren que L290 estaba presente en la rodopsina ancestrales inferidas, en concordancia con las teorías del ?cuello de botella nocturno?en mamíferos. La substitución L290I podría haber tenido un importante papel en la adaptación y la evolución molecular de las rodopsina de mamíferos al ser probablemente el resultado de cambios análogos independientes, hecho que puede ser apreciado en los órdenes de primates y roedores. Esta hipótesis fue confirmada experimentalmente mediante la mutación L290I en rodopsina murina que resultó en una tasa de decaimiento del Meta II similar al de, Postprint (published version)

Peer Reviewed, Postprint (published version)

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments, Peer Reviewed, Postprint (author's final draft)

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors., Peer Reviewed, Postprint (published version)

Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa., Peer Reviewed, Postprint (author's final draft)

El resveratrol (3, 5,4’-trihidroxi-trans-estilbeno) és un polifenol no flavonoide que està present en diversos aliments com a raïms, nabius, cacauets, etc. Mitjançant diverses recerques s'ha demostrat que el resveratrol exerceix un paper anti-oxidant i anti inflamatorio, cardio-protector, quimioterapéutico i neuroprotector en diverses malalties a causa dels seus molts mecanismes d'acció. Aquest efecte ha generat interès de la comunitat científica i s'han realitzat recerques in vitro com in vivo en animals i éssers humans. Malgrat totes les recerques realitzades, no hi ha estudis concloents de com el resveratrol beneficia a les persones, quins són els seus efectes adversos, quines interaccions presenta amb altres medicaments i què determina la baixa biodisponibilidad en éssers humans. Sent aquesta última una de les limitacions més importants, s'ha investigat diferents alternatives per millorar la seva absorció. Una altra característica destacable del resveratrol és el seu ràpid metabolisme, és absorbit ràpidament per les cèl·lules epitelials intestinals i hepatòcits, la qual cosa dificulta l'absorció i arribada als teixits i sang. En les recerques sobre les malalties oculars, la majoria van concloure de manera positiva sobre l'efecte del resveratrol malgrat no ser afirmacions concloents. Coincidien que el resveratrol controla la progressió de les patologies, fins i tot ha arribat a disminuir els signes i símptomes d'aquestes malalties i evitar la seva aparició.

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily., Peer Reviewed, Postprint (author's final draft)

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, Los polifenoles de la dieta representan, un grupo de metabolitos secundarios los cuales se encuentra ampliamente en frutas y vegetales, vino, te, aceite de oliva extra virgen, chocolate y otros productos de cacao. Estudios previos han mostrado la interacción de algunos compuestos fenólicos con receptores de estrógeno y adenosina, pertenecientes a la superfamilia de receptores acoplados a proteína G (GPCRs). Estos GPCRs son la familia más grande de moléculas de transducción de señales involucradas en la mayoría de procesos fisiológicos. Rodopsina es el miembro prototípico de GPCRs y es la proteína mayoritaria encontrada en la membrana de los discos del segmento exterior de los bastones, células fotoreceptoras de la retina, y el primer con la estructura cristalina resuelta. Mutaciones en rodopsina se han asociado con retinosis pigmentaria (RP), un grupo de enfermedades visuales hereditarias que causan degeneración retiniana la cual lleva a la pérdida progresiva de las células fotoreceptoras, bastones y conos. Causando ceguera. Numerosos estudios sobre mutantes de RP se han llevado a cabo con la finalidad de elucidar los mecanismos moleculares de la enfermedad como un primer paso necesario antes de que se puedan desarrollar enfoques terapéuticos adecuados. Algunos de los tratamientos propuestos se basan en el rescate farmacológico, en el cual moléculas pequeñas conocidas como chaperonas químicas o farmacológicas se unen y estabilizan opsinas mal plegadas. Los polifenoles se han propuesto como agentes útiles contra la toxicidad retiniana, pero hasta ahora no se ha presentado evidencia clara de los efectos de estos compuestos a nivel molecular del sistema de fototransducción visual. Dado el interés en encontrar nuevos ligandos que puedan compensar los efectos causados por mutaciones de RP, el objetivo de esta tesis fue evaluar el efecto de los polifenoles sobre la estructura y función la rodopsina y mutantes de RP G90V, Y102H e 1307N y estudiar las preferencias de unión, Postprint (published version)

G-protein coupled receptors (GPCRs) are the largest membrane protein superfamily encoded by the human genome and represent the largest class of drug targets for a wide range of pathological conditions. Two GPCRs members, the visual pigment rhodopsin associated with the retinal degenerative disease retinitis pigmentosa (RP), and the muscarinic acetylcholine (ACh) 3 receptor (M3R) associated with Alzheimer’s disease (AD), are studied in this thesis. Rhodopsin is the prototypical visual photoreceptor mediating scotopic vision and distributed throughout the retina. Upon illumination, the bound chromophore 11-cis-retinal isomerizes to all-trans-retinal and triggers the visual signaling cascade. Mutations found in rhodopsin, such as G90V and N55K, are responsible for the retinal degenerative disease RP. To counteract the low structural stability of these mutants, and to provide a deeper understanding of the molecular mechanisms leading to visual dysfunction, artificial membranes in the form of DMPC/DHPC bicelles and DDHA-PC liposomes were prepared. DMPC/DHPC bicelles and DDHA-PC liposomes provided a native-like bilayer environment, which preserved rhodopsin wild type and mutants structure and increased their thermal stability to varying degrees compared to the usual dodecyl maltoside (DM) detergent. Furthermore, chromophore regeneration of G90V and N55K mutants in DMPC/DHPC bicelles condition was enhanced compared to DM condition. Moreover, the kinetics for the active state metarhodopsin II (Meta II) decay indicated that retinal release rates of G90V and N55K mutants became faster in the presence of DMPC/DHPC bicelles and DDHA-PC liposomes compared to DM condition. The addition of hydroxylamine upon Meta II complete decay of WT and G90V in bicelles increased fluorescence intensity, suggesting that retinal can be retained inside the binding pocket. DMPC/DHPC bicelles and DDHA-PC liposomes provided stable conditions so that G90V opsin, obtained after Meta II completely deca, Los receptores acoplados a proteína G (GPCRs) representan la mayor superfamilia de proteínas de membrana codificada por el genoma humano y también la mayor clase de dianas terapéuticas para diversas enfermedades. En esta tesis se estudian dos miembros de los GPCRs; el pigmento visual rodopsina, asociado con la enfermedad degenerativa de la retina retinitis pigmentosa (RP) y el receptor de acetilcolina (ACh) muscarínico 3 (M3R) asociado con la enfermedad de Alzheimer (AD). La rodopsina es el fotorreceptor visual prototípico responsable de la visión escotópica y se encuentra distribuido por toda la retina. Después de la iluminación, el cromóforo 11-cis-retinal se isomeriza a todo-trans-retinal y desencadena serie de reacciones intracelulares llegando al nervio óptico y permitiendo la unión. Mutaciones en rodopsina, tales como G90V y N55K son causantes de RP. Para entender más profundamente los mecanismos moleculares causantes de esta disfunción visual debido a estas mutaciones, se han preparado membranas artificiales tales como bicelas de DMPC/DHPC y liposomas de DDHA-PC. Estos sistemas lipídicos ofrecen un entorno bicapa más nativo, comparado con el que proporciona el detergente dodecil maltosido (DM), usado más tradicionalmente, lo que preserva la estructura de la rodopsina nativa y de los mutantes y aumenta su estabilidad térmica. La regeneración cromóforo para de G90V y N55K en bicelas de DMPC/DHPC es más elevada en comparación con el valor obtenido en DM. La cinética de decaimiento de la conformación activa metarodopsina II (Meta II) indica que la velocidad de liberación del retinal de los mutantes G90V y N55K es más alta en presencia de las bicelas de DMPC/DHPC, y de los liposomas de DDHA-PC, en comparación con las velocidades obtenidas en DM. La adición del reactivo hidroxilamina después del decaimiento completo de Meta II de WT y del mutante G90V en las bicelas provoca un incremento de la intensidad de fluorescencia, indicando que parte de Meta II aún mantiene, Postprint (published version)

La rodopsina es un fotopigmento, concretamente una glicoproteína integral de membrana que pertenece a la familia de los receptores acoplados a proteínas G (GPCR). Tiene la función de detectar los fotones y responder a un estímulo luminoso. A partir del experimento realizado en el que se ha utilizado rodopsina bovina y ha sido iluminada directamente bajo diferentes intensidades en periodos de tiempo medidos se ha podido comprobar que en un periodo corto de tiempo (5s) se puede llegar prácticamente al blanqueamiento total del fotorreceptor. En cambio cuando ésta es sometida a una intensidad de iluminación mínima la cantidad de absorbancia va disminuyendo progresivamente, este comportamiento confirma su función de fotorreceptor que trabaja en condiciones de baja iluminación.

Informe cientític del projecte realitzat per a la Fundación Ramón Areces, Las mutaciones en la rodopsina son la principal causa de retinosis pigmentaria (RP) autosómica dominante. Estas mutaciones pueden clasificarse según sus propiedades bioquímicas y celulares. Por ejemplo las de tipo II son las más comunes y causan mal plegamiento estructural de la proteína. El plegamiento, la degradación y la agregación de algunas de estas rodopsinas mutadas pueden ser modificados mediante fármacos y chaperonas moleculares. Asimismo, mutaciones en las opsinas de las células fotorreceptoras cono causan distrofia de conos y bastones. El principal objectivo de este proyecto es entender el(los) mecanismo(s) molecular(es) del mal plegamiento de la rodopsina, de las opsinas de los conos, inducido por mutaciones genéticas y verificar la hipótesis de que algunos fármacos y chaperonas moleculares pueden afectar el destino de las opsinas mutadas. Se determinará el efecto de mutaciones de RP y de distrofia de conos y bastones en la estructura y función de la proteínas mutadas expresadas en cultivos celulares. Se utilizarán retinales sintéticos y zinc como dos de los factores para modular la expresión y el plegamiento de las opsinas mutadas. Se utilizarán modelos celulares establecidos para determinar el efecto de fármacos inductores de chaperonas moleculares como agentes frente a las propiedades tóxicas de ganancia de función de las opsinas mutadas. Finalmente, se estudiarán las chaperonas que pueden modular el plegamiento, la degradación y el destino celular y la función de las opsinas mutadas mediante la sobreexpresión y RNAi. Estos estudios aumentarán nuestros conocimientos sobre la biología celular retiniana fundamental y sobre la biología de los receptores acoplados a proteínas G, superfamilia a la que pertenecen la rodopsina y los pigmentos de los conos, en particular. Aún más importante será el hecho de que los resultados obtenidos deberán ayudar a desarrollar nuevos tratamientos para la RP así como para la distrofia de conos y bastones causadas por mutac, Preprint

The final publication is available at Springer via http://dx.doi.org/10.1007/s00018-015-2043-7, Human red and green visual pigments are seven transmembrane receptors of cone photoreceptor cells of the retina that mediate color vision. These pigments share a very high degree of homology and have been assumed to feature analogous structural and functional properties. We report on a different regeneration mechanism among red and green cone opsins with retinal analogs using UV–Vis/fluorescence spectroscopic analyses, molecular modeling and site-directed mutagenesis. We find that photoactivated green cone opsin adopts a transient conformation which regenerates via an unprotonated Schiff base linkage with its natural chromophore, whereas red cone opsin forms a typical protonated Schiff base. The chromophore regeneration kinetics is consistent with a secondary retinal uptake by the cone pigments. Overall, our findings reveal, for the first time, structural differences in the photoactivated conformation between red and green cone pigments that may be linked to their molecular evolution, and support the proposal of secondary retinal binding to visual pigments, in addition to binding to the canonical primary site, which may serve as a regulatory mechanism of dark adaptation in the phototransduction process., Peer Reviewed, Postprint (author's final draft)

Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. 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. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution., Peer Reviewed, Postprint (published version)

This is the peer reviewed version of the following article: A. Peralvárez-Marín, P. Garriga, Optogenetics Comes of Age: Novel Inhibitory Light-Gated Anionic Channels Allow Efficient Silencing of Neural Function. ChemBioChem 2016, 17, 204-206, which has been published in final form at http://dx.doi.org/10.1002/cbic.201500608. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving, Optogenetics, the developing field of research that uses light-switchable biochemical tools in a sophisticated technological approach to monitor or control neural function, is rapidly evolving with the discovery and development of novel microbial rhodopsins. Light-absorbing membrane proteins, as tools for brain research, are promoting new applications within the discipline of optogenetics. Light-gated rhodopsin ion channels with better intrinsic light sensitivity and improved resolution are needed to overcome some of the current limitations of existing molecules. The recent discovery of light-gated inhibitory anion channels opens new opportunities for studying physiological neural processes and, at the same time, represent a powerful approach for elucidating the mechanisms of neurological and mental disorders that could benefit from this approach., Peer Reviewed, Postprint (published version)

The final publication is available at Springer via http://dx.doi.org/10.1007/s12035-015-9153-y, 5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease., Peer Reviewed, Postprint (author’s final draft)

Human color perception is mediated by cone photoreceptor cells which mainly locate on the fovea of the eye. Bright light activates the photosensitive opsin pigments which are embedded in the outer segment membrane discs of cone retinal cells thereby initiating the complex process of photopic vision with a fast response. The cone visual pigments are G-protein coupled receptors which share analogous structure and functional features with rhodopsin, the most thoroughly studied G-protein coupled receptor from rod photoreceptor cells mediating scotopic vision and distributed throughout the retina. These visual pigments modulate spectral tuning of visible light depending on the molecular variance around the protein bound chromophore, 11-cis-retinal, which is a vitamin A derivative acting as an inverse agonist. Various mutations have been clinically identified in the cone opsin genes and associated with visual dysfunction ranging from mild color blindness to severe cone dystrophies. As the crystal structure of the cone pigments is yet to be resolved, identifying key molecular mechanisms that play major roles in optimal functioning of these photoreceptor proteins, should be helpful in providing a deeper understanding of their function and in designing novel therapeutic strategies for congenital retinal cone dysfunction. In order to study such light sensitive, delicate membrane proteins, the human cone opsin genes have been transiently expressed in mammalian cells, regenerated with their natural chromophore and immunopurified in dark conditions. The purified recombinant chromophore-regenerated cone opsins in solution have been characterized in detail by means of biophysical approaches, including spectroscopic, biochemical, and functional analysis, in order to uncover novel properties that help in optimizing the function of these receptors. Site-directed mutagenesis was employed to obtain the clinically identified mutations in cone opsins related to visual disorders and to co, La percepción del color humano se realiza a través de las células fotorreceptoras de los conos, localizados en la fóvea del ojo. La luz brillante activa los pigmentos fotosensibles que se encuentran en los discos de membrana del segmento externo de estas células iniciando con ello el complejo y rápido proceso de la visión fotópica. Los pigmentos visuales conos son receptores acoplados a proteínas G (GPCR) que comparten estructura análoga y características funcionales a la rodopsina, el GPCR más estudiado y localizado en las células fotorreceptoras de los bastones y responsable de la visión escotópica . Estos pigmentos visuales modulan la sintonización espectral de la luz visible, dependiendo de las diferencias moleculares alrededor del cromóforo ligado a la proteína, 11-cis-retinal, que es un derivado de la vitamina A y que actúa como un agonista inverso. Varias mutaciones han sido identificadas clínicamente en los genes de las opsinas de los conos asociadas a disfunciones visuales; desde la leve ceguera al color a la grave distrofia de las células fotorreceptoras. Como la estructura cristalina de los pigmentos de los conos está todavía por resolver, identificar mecanismos moleculares que juegan un papel importante en el funcionamiento óptimo de estas proteínas, ayudará a obtener una comprensión más profunda de su función y al diseño de nuevas estrategias terapéuticas para la disfunción congénita de los conos. Con el fin de estudiar estas proteínas de membrana sensibles a la luz y delicadas, se han expresado transitoriamente en células de mamífero, regenerado con su cromóforo natural, inmunopurificado en oscuridad y se han caracterizado por medio de técnicas biofísicas, bioquímicas, y funcionales, a fin de descubrir nuevas propiedades que ayuden a la optimización de su función. Mutagénesis dirigida se ha utilizado para obtener las mutaciones clínicamente identificados en opsinas de conos relacionados con los trastornos visuales y comparar la estructura y la función, Postprint (published version)

Rhodopsin is a glycoprotein formed by a proteic part (opsin) and a non - proteic part (11 - cis - retinal), which is located in the rod outer segment discs . Rods are one of the two kinds of photoreceptors found in the retina, which make vision possible at low light conditions. Retinitis Pigmentosa (RP) are a group of degenerative diseases caused by alterations in rhodopsinstructure, due to genetic mutations. These diseases directly affect night vision and the visual field. Despite an efficient treatment has not been found yet, a recent study (Mahesh et al., 2012) showed that RP patients treated with valproi c acid during a 3 month period increase d their visua l acuity and their visual field. In the present study, this acid has been used as a sodium salt form (sodium valproate), and different parameters have been determined in vitro , including rhodopsin therm al stability at different temperatures and different concentrations; rhodopsin regeneration; rhodopsin behaviour to wards acidification; and rhodopsin aromatic amino acid s fluorescence. All these parameters have been measured in the presence and in the absence of sodium valproate. The results obtained show th at sodium valproate reduces rhodopsin regeneration time, a s well as the regeneration percentage . Also, due to sodium valproate, rhodopsin releases the retinal faster in fluorescence tests. In contrast , it does not a ffect in such a significant way rhodopsin therm al stability and its behaviour to acidification., La rodopsina es una glicoproteína formada por una parte proteica (opsina) y una parte no proteica (11 - cis - retinal) . S e localiza e n los discos de los segmentos externos de los bastones, uno de los 2 tip o s de fotoreceptores que se encuentran en la retina y hacen posible la visión con poca luz. Las retinosis pigmentarias (RP), son enfermedades degenerativas causadas por alteraciones e n la estructura de la rodopsina debidas a mutaciones genéticas. Estas enfermedades se caracterizan por la afectación en la visión nocturna y el campo visual. Actualmente, todavía no se ha encontrado un tratamiento efectivo , pero un estudio reciente (Mahes h et al., 2012) indica que la administración de ácido valproico durante 3 meses a pacientes con retinosis pigmentarias les ha hecho aumentar la agudeza visual y el campo visual. En el presente estudio se ha trabajado con este ácido en forma de sal sódica ( valproato sódico ), y se ha determinado in vitro la estabilidad térmica de la rodopsina a diferentes temperaturas y concentraciones , así c omo la regeneración de la proteína , su comportamiento al acidificar y la posible variación en la fluorescencia de los aminoácidos aromáticos presentes en la rodopsina, tanto en ausencia como en presencia de valproato sódico . Los resultados obtenidos muestran que el valproato sódico causa una regeneración más rápida y en un mayor porcentaje de la rodopsina, además de hace r que ésta libere más rápidamente el retinal en los experimentos de fluorescencia, aunque no influye de manera tan significativa en la estabilidad térmica y la acidificación de la rodopsina ., La rodopsina és una glicoproteïna formada per una part proteica (opsina) i una part no proteica (11 - cis - retinal). E s localitza als discs del s segments externs dels bastons, un dels 2 tipus de fotoreceptors que es troben a la retina i que fan possible la visió a mb poca llum. Les retinosis pigmentàries (RP) són malalties degeneratives causades per alteracions en l’estructura de la rodopsina degudes a mutacions genètiques. Aquestes malalties es caracteritzen per l’afectació en la visió nocturna i el camp visual. A ctualment , encara no s’ha trobat un tractament efectiu , però un estudi recent (Mahesh et al., 2012) indica que l’administració d ’ àcid valproic durant 3 mesos a pacients amb RP el s ha fet augmentar l’ agudesa visual i el camp visual . En el present estudi s ’ha treballat amb l’ àcid valproic en forma de sal sòdica ( valproat de sodi ), i s’ha determinat in vitro l’estabilitat tèrmica de la rodopsina a diferents temperatures i concentracions , així com la regeneració de la proteïna , el seu comportament a l ’ acidi ficar i la possible variació en la fluorescència dels aminoàcids aromàtics presents a la rodopsina , tant en abs ència com en presència de valproat de sodi . Els resultats obtinguts mostren que el valproat de sodi fa que la rodopsina regeneri més ràpid i en un percentatge més alt , i alliberi més ràpid el retinal en els experiments de fluorescència, tot i que no influeix de manera tan significativa en l ’estabilitat tèrmica i el comportament a l’ acidifica r .

Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, NSSK and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC 0 50 100 bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes., Postprint (published version)

GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor-receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR(1) which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR(1)-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions.; We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR(1) and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric-homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR(1) shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels. (C) 2015 Elsevier B.V. All rights reserved., Peer Reviewed, Postprint (author's final draft)

The type of serotonin receptor 1A (5-HT1A), the galanin type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily The serotonin receptor type 1A (5-HT1A), the galanin receptor type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily of G-protein-coupled receptors. All three GPCRs share, among other features, a relationship with the pathophysiology of unipolar depression associated with presence of zinc. The current paradigm with respect to these functional unities is that these receptors are not acting as monomeric forms but through complexs involving specific interactions with themselves or other receptors from the same family. This allows them to increase their functional possibilities exponentially, allowing versatility from a fixed number of receptors, and consequently also increases the number of pharmacological approachmes. The heterodimerization between 5-HT1A receptor and GalR1 has been previously described as an antagonistic interaction that could lead to unipolar depression. In this thesis we have shown that this interaction is avoided in the presence of zinc, giving a rational explanation for the antidepressant effect widely described for this cation. Although there was no published evidence regarding the interaction of these two receptors with GPR39, a receptor that is activated by zinc and whose expression depends on the concentration of this cation, in this thesis we have demonstrated the ability to interact of these three receptors in both forms GPR39-5-HT1A, 5-HT1A-GalR1 and the trimer GPR39-5-HT1A-GalR1. It has also been found that the functional capacity of the receptors is modified according the type of interaction in which these receptors are involved. So that monomeric and oligomeric forms have different signalling capacities. This would suggest that in the human brain all putative receptor configurations could be present, and that the presence of one or other would be regulated by zinc concentration. Deepeni, El receptor de serotonina del tipus 1A (5-HT1A), el de galanina de tipus 1 (GalR1) i el receptor orfe 39 (GPR39) pertanyen a la superfamília dels receptors acoblats a proteïna G. Tots tres receptors comparteixen, entre d'altres característiques, una relació amb la fisiopatologia de la depressió unipolar associada amb la presència de zinc. El paradigma actual, respecte a la unitat funcional d'aquests receptors, és que no actuen en forma monomèrica sinó mitjançant complexos que involucren interaccions específiques amb ells mateixos o amb altres receptors de la mateixa família. Això els hi permet augmentar les seves possibilitats funcionals exponencialment, permetent una gran versatilitat a partir d'un nombre fix de receptors, i en conseqüència també augmenta el nombre de possibilitats d'abordatge farmacològic. L'heterodimerització entre el receptor 5-HT1A i el GalR1 s'ha descrit prèviament com una interacció antagònica que podria donar lloc a la depressió unipolar. En aquesta tesi s'ha demostrat que aquesta interacció s'evita en presència de zinc, donant una explicació racional a l'efecte antidepressiu àmpliament descrit per a aquest catió. Tot i que no hi havia cap evidència publicada respecte a la interacció d'aquests dos receptors amb el GPR39, un receptor que és activat pel zinc i l'expressió del qual depèn de la concentració del mateix, en aquesta tesi s'ha demostrat la capacitat d'interacció d'aquests tres receptors, tant en les formes GPR39-5-HT1A i 5-HT1A-GalR1 com en el trímer GPR39-5-HT1A-GalR1. A més, s'ha trobat que la capacitat funcional dels receptors es veu modificada segons el tipus d'interacció en la que aquests receptors participen, de manera que les formes monomèriques i oligomèriques presenten una capacitat de senyalització diferent. Això faria pensar que en el cervell humà podrien trobar-se totes les configuracions potencials de receptors, i que la presència d'unes o altres estaria regulada per la concentració de zinc. L'aprofundiment en el mecani, Postprint (published version)

En este proyecto se ha estudiado la posible interacción de la rodopsina con dos compuestos fenólicos: un flavonoide, la catequina y un ácido fenólico, el ácido cafeico. Los fenoles y flavonoides son sustancias que se encuentran en alimentos de nuestra dieta diaria como las frutas y las verduras. Proporcionan colores a las plantas y las protegen de los rayos UV. Tienen propiedades antioxidantes, antiinflamatorias, antivirales, y antifúngicas que los hacen beneficiosos para nuestra salud, pudiendo prevenir enfermedades degenerativas y trastornos de la edad, así como también inhibir el crecimiento de células cancerígenas, entre otros efectos. Respecto a la salud ocular, la catequina podría prevenir la formación de cataratas, protegiendo al cristalino de los rayos UV, y por otro lado la actividad antiangiogénica del ácido cafeico podría proteger frente a patologías retinianas como la DMAE y la retinopatía diabética. En este estudio se han utilizado muestras de rodopsina solubilizada, extraída de retinas bovinas, hidrato de catequina y ácido cafeico para realizar experimentos in vitro de estabilidad térmica y de regeneración del pigmento visual rodopsina. Mediante espectrofotometría de absorción UV-visible se ha podido determinar que el ácido cafeico puede aumentar la estabilidad de la rodopsina, y tanto la catequina como el ácido cafeico mejorarían la regeneración de la rodopsina a partir de opsina y retinal. Estos resultados sugieren un papel beneficioso de estos compuestos a nivel molecular de las células fotorreceptoras. Se requerirían estudios adicionales para confirmar estos efectos, y su posible extrapolación in vivo., En aquest projecte, s’ha estudiat la possible interacció de la rodopsina amb dos compostos fenòlics: un flavonoide, la catequina i un àcid fenòlic, l’àcid cafeic. Els fenols i flavonoides són substancies que es troben en aliments de la nostra dieta diària com les fruites i les verdures. Proporcionen colors a les plantes i les protegeixen dels raigs UV. Tenen propietats antioxidants, antiinflamatòries, antivirals, i antifúngiques que els fan beneficiosos per a la nostra salut, prevenint malalties degeneratives i trastorns de l’edat, així com també inhibir el creixement de cèl·lules cancerígenes, entre d’altres efectes. Respecte a la salut ocular, la catequina podria prevenir la formació de cataractes, protegint al cristal·lí del raig UV, i per un altre banda l’activitat antiangiogènica de l’àcid cafeic podria protegir en front a patologies retinianes com la DMAE i la retinopatia diabètica. En aquest estudi s’han utilitzat mostres de rodopsina solubilitzada, extreta de retines bovines, hidrat de catequina i àcid cafeic per realitzar experiments in vitro d’estabilitat tèrmica i de regeneració del pigment visual rodopsina. Mitjançant espectrofotometria d’absorció UV-visible s’ha pogut determinar que l’àcid cafeic pot augmentar l’estabilitat de la rodopsina, i tant la catequina com l’àcid cafeic millorarien la regeneració de la rodopsina a partir d’opsina i retinal. Aquest resultats suggereixen un paper beneficiós d’aquests compostos a nivell molecular de les cèl·lules fotoreceptores. Es requeririen estudis addicionals per confirmar aquests efectes, i la seva possible extrapolació in vivo., In this project we have studied the possible interaction of rhodopsin with two phenolic compounds: a flavonoid such as catechin and a phenolic derivative like caffeic acid. Phenols and flavonoids are found in foods really common in our daily diet like fruits and vegetables. They provide color to plants and protect them from UV rays at the same time. Flavonoids are known to have antioxidant, anti-inflammatory, antifungal and antiviral properties, which make them beneficial to human health. Phenolic compounds are associated with a reduced risk of major agerelated diseases, as well as inhibition of the growth of cancer cells, among other effects. In visual health, catechin could prevent the formation of cataracts protecting the crystallin lens against UV rays. On the other side, the antiangiogenic activity of caffeic acid could protect us against retinal diseases like AMD and diabetic retinopathy. In this study, we used solubilized rhodopsin samples extracted from bovine retinas, catechin and caffeic acid to perform in vitro experiments on the thermal stability and regeneration of the rhodopsin visual pigment. By means of UV-visible absorption spectrophotometry it has been found that caffeic acid can increase the stability of rhodopsin, and both catechin and caffeic acid would improve the regeneration of rhodopsin from opsin and retinal. These results suggest a beneficial role of these compounds at the molecular level of the photoreceptor cells. Additional studies are needed to confirm these effects, and their possible in vivo extrapolation.

In recent years, important advances have been made to decipher the multiple defense mechanisms of the retina (a highly specialized sense tissue) against several extreme conditions, such as constant exposure to systemic toxins, oxidative stress, and focused light rays. These studies have importantly contributed to improving our understanding of the principles underlying various drug- and light-induced disease processes, specifically at the eye level. When the different retinal cell layer defenses are overwhelmed by various xenobiotics, environmental agents such as pollution, cigarette smoke, or excessive light exposure, particularly high-frequency blue light and ultraviolet light, a pathological process may develop. Herein, we describe the main aspects of the structure and function of the retina in connection to some of the most relevant mechanisms which may generate retinal toxicity, and possible alternatives to counteract these effects. Furthermore, we summarize current knowledge about light-induced retinal toxicity as well as the effects of different natural and synthetic compounds on the molecular mechanisms underlying toxicity in the retina. Finally, the potential approaches undertaken to counteract these toxic effects, i.e., polyphenols and others, are discussed., Peer Reviewed, Postprint (published version)

Recientemente se han logrado grandes avances en la determinación de la estructura y función de los receptores acoplados a proteínas G (GPCR’s). Los GPCR´s constituyen gran familia de proteína integrales de membrana y se encuentran implicados en una gran variedad de procesos fisiológicos como la respuesta a hormonas y neurotransmisores, así como también son centrales en los sistemas sensoriales mediando las respuestas en los sentidos de la vista, olfato y el gusto. Entre ellos se encuentra la familia de los receptores muscarínicos de acetilcolina, compuesta por 5 subtipos de receptores (M1-M5), los cuales modulan la mayor parte de las acciones del neurotransmisor acetilcolina en el sistema nervioso central y en el sistema nervioso periférico. Los receptores muscarínicos han jugado un papel muy relevante en la compresión del funcionamiento de la neurotransmisión. En este trabajo nos centraremos únicamente en los receptores M1 y M3 debido a su potencial participación en el mecanismo molecular de la enfermedad de Alzheimer Hasta ahora se ha visto que estos receptores se encuentran implicados en varias patologías del sistema nervioso central, ya que modulan la señalización de diferentes sistemas neurotransmisores. Estos sistemas se ven alterados como en el caso de la enfermedad de Alzheimer, donde se ha visto que modulan la degradación de la APP (proteína precursora de amiloide) por la α-secretasa y de esta forma evitan la formación de las placas neuríticas conocidas como β-amiloides (Aβ) características de esta enfermedad. Además se ha sugerido que como resultado de su interacción con la proteína tau, pueden jugar un papel muy relevante en la pérdida de neuronas colinérgicas a través de un mecanismo que involucra la interacción intracelular y extracelular de la proteína tau con los receptores muscarínicos M1/M3 presentes en este tipo de neuronas. El objetivo de este trabajo es comprender y analizar detalladamente las propiedades biológicas y bioquímicas de las diferente

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1-15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1-GalR2 heteromers which may play a significant role in mediating galanin fragment (1-15) signaling. In HEK293T cells evidence for the existence of GalR1-GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET2 assays. PLA positive blobs representing GalR1-GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1-15) was more potent than galanin (1-29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1-GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1-15) and of galanin (1-29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (115) vs galanin (1-29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1-29). In contrast, in NFAT reporter gene assays galanin (1-29) shows a higher efficacy than galanin (115) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1-GalR2 heteroreceptor complexes induced by galanin (1-15) may contribute to depression-like actions since GalR1 agonists produce such effects. (C) 2014 Elsevier Inc. All rights reserved., Postprint (published version)

El procés visual s’inicia quan els fotons incideixen sobre els fotoreceptors localitzats a la retina i l’energia electromagnètica continguda en ells es transforma en un impuls nerviós. La retina dels vertebrats té dos tipus de fotoreceptors, cons i bastons, cadascun d’ells amb una proteïna capaç de realitzar aquesta transformació, conopsines i rodopsina respectivament; en aquest estudi hem triat la que es troba en els bastons, la rodopsina. Els oligoelements, coneguts també com a elements traça, són aquells que l’ésser humà no pot sintetitzar per si mateix, de manera que els ha d’obtenir de la dieta. La seva presència es requereix en quantitats específiques, així la seva absència és perjudicial per la salut, però un excés pot ser igual de dolent. Els oligoelements estudiats han estat: coure, manganès, níquel, crom i ferro. Aquests elements habitualment es troben en suplements alimentaris i s’acumulen en diferents òrgans entre ells els ulls, i poden alterar la seva funció. En aquest estudi s’ha determinat l’estabilitat tèrmica a 55º C i la capacitat de regeneració de la rodopsina tant en presència com en absència d’aquests bioelements. Els resultats obtinguts confirmen la hipòtesis de que els bioelements influeixen, en major o menor grau tant en l’estabilitat tèrmica de la rodopsina com en la seva capacitat de regeneració.

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin imrnunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light., Peer Reviewed, Postprint (published version)

The molecular chaperone Hsp90 is important for the functional maturation of many client proteins, and inhibitors are in clinical trials for multiple indications in cancer. Hsp90 inhibition activates the heat shock response and can improve viability in a cell model of the P23H misfolding mutation in rhodopsin that causes autosomal dominant retinitis pigmentosa (adRP). Here, we show that a single low dose of the Hsp90 inhibitor HSP990 enhanced visual function and delayed photoreceptor degeneration in a P23H transgenic rat model. This was associated with the induction of heat shock protein expression and reduced rhodopsin aggregation. We then investigated the effect of Hsp90 inhibition on a different type of rod opsin mutant, R135L, which is hyperphosphorylated, binds arrestin and disrupts vesicular traffic. Hsp90 inhibition with 17-AAG reduced the intracellular accumulation of R135L and abolished arrestin binding in cells. Hsf-1(/) cells revealed that the effect of 17-AAG on P23H aggregation was dependent on HSF-1, whereas the effect on R135L was HSF-1 independent. Instead, the effect on R135L was mediated by a requirement of Hsp90 for rhodopsin kinase (GRK1) maturation and function. Importantly, Hsp90 inhibition restored R135L rod opsin localization to wild-type (WT) phenotype in vivo in rat retina. Prolonged Hsp90 inhibition with HSP990 in vivo led to a posttranslational reduction in GRK1 and phosphodiesterase (PDE6) protein levels, identifying them as Hsp90 clients. These data suggest that Hsp90 represents a potential therapeutic target for different types of rhodopsin adRP through distinct mechanisms, but also indicate that sustained Hsp90 inhibition might adversely affect visual function., Peer Reviewed, Postprint (published version)