The first event in light perception is absorption of a photon by an opsin visual pigment in a rod or cone photoreceptor cell. This causes isomerization of the 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which decays to yield apo-opsin and free all-trans-RAL. Light sensitivity is regained by apo-opsin when it recombines with another 11-cis-RAL. Synthesis of 11-cis-RAL is carried out by an enzyme pathway called the Visual Cycle in cells of the retinal pigment epithelium (RPE). Accumulating evidence suggests that cones may have access to another source of visual chromophore in M?ller cells through a second hypothesized pathway called the Alternate Visual Cycle. This pathway is thought to provide visual chromophore specifically to cones under daylight conditions. We identified the critical isomerase of the Alternate Visual Cycle (isomerase-2) as dihydroceramide desaturase-1 (DES1). The rate of retinol isomerization by DES1 is 300-fold faster than the rate of isomerization catalyzed by Rpe65 of the RPE Visual Cycle. Clinically, the Alternate Visual Cycle, which is located entirely within the retina, may protect photoreceptors from rapid degeneration following retinal detachment, where the retina and RPE become physically separated. This proposal is to characterize the visual-retinoid processing activity of DES1, and to study its role in the Alternate Visual cycle. We recently made the important preliminary observation that the isomerase-2 activity of DES1 is potently stimulated by visible light. This result suggests that under daylight conditions, vertebrates (including humans) capture light energy in their retinas to regenerate visual chromophore. It has been known for some time that insects and other invertebrates regenerate their visual pigments using light energy. The observation that light contributes to chromophore regeneration in animals is unprecedented. Further, this process appears to take place through a novel biochemical mechanism: absorption of a 550-nm photon by a radical-cation of vitamin A inside the DES1 protein. Besides in-depth biochemical characterization of DES1, we will study light-stimulated regeneration of visual chromophore in cultured cells and in live, genetically modified mice.
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