New Mechanisms for Regeneration of Visual Pigments in the Vertebrate Retina
Vision begins when a photon is captured by a visual pigment in a photoreceptor cell. For most vertebrates, the light-sensitive chromophore in the visual pigment is 11-cis-retinaldehyde (11-cis-retinal), which is covalently bound to the protein moiety, opsin, through a Schiff base linkage. The absorption of a photon isomerizes 11-cis-retinal to its all-trans-isomer and activates the visual pigment. In vertebrate ciliary photoreceptors, after a brief activation, the pigment decays to yield apo-opsin and free all-trans-retinal. Light sensitivity restoration in photoreceptor cells occurs following re-isomerization of all-trans-retinal to 11-cis-retinal by enzymatic pathways termed “visual cycles”. At present, two visual cycles have been established: (1) A well understood canonical visual cycle between RPE cells and photoreceptors, with RPE65 as its retinoid isomerase (Isomerase I); (2) A relatively under characterized alternate visual cycle between Mï¿½ller cells and cones, with an unknown retinoid isomerase (Isomerase II). Recently, we have identified dihydroceramide desaturase-1 (DES1) as a novel retinoid isomerase and a strong candidate for Isomerase II. In this dissertation, follow-up biochemical characterizations of DES1 were performed. Our results suggest that DES1 coimmunoprecipitates with cellular retinaldehyde-binding protein (CRALBP). Its retinoid isomerase activity appears to (1) rely on the cytochrome b5 electron transport chain, (2) involve a free radical intermediate(s) and (3) an iron (iii) co-factor (Chapter 2.1). A Des1-/- mouse model was also investigated. Although neither obvious retina morphological abnormality nor significant reduction of visual chromophore after overnight dark-adaptation was observed in Des1-/- mice, the key point, their ability to recover cone sensitivity after bleach, has not been tested. Further studies are needed to address the physiological role of DES1 in the alternate visual cycle (Chapter 2.2). Furthermore, attempts were made to identify a putative retinol dehydrogenase in cones that enable them to utilize the alternate visual cycle for pigment regeneration exclusively. But this enzyme remains unidentified despite our efforts (Chapter 2.3). Besides the two known enzymatic visual cycles, we also identified a novel non-enzymatic photoisomerization pathway for functional visual pigment regeneration in the vertebrate retina with a photoactive intermediate, N-retinylidene-phosphatidylethanolamine (N-ret-PE) (Chapter 3). Our findings greatly promote the current understanding of visual pigment regeneration mechanisms.