These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.
Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the alltrans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of -carotene dioxygenase, appears to play a role in this pathway. Rpe65 ؊/؊ knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds alltrans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65 ؊/؊ mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.Light perception in vertebrates is mediated by a group of G protein-coupled receptors called the opsins. Most opsin pigments contain 11-cis-retinaldehyde (11cRAL) 1 as the light-absorbing chromophore. Absorption of a photon induces 11-cis to all-trans isomerization of the chromophore, resulting in the activated species, metarhodopsin II. After a brief period, metarhodopsin II decays to yield apo-rhodopsin and free alltrans-retinaldehyde (atRAL). Before light sensitivity of the pigment can be restored, the atRAL must be chemically re-isomerized to 11cRAL by a metabolic pathway called the visual cycle. Most steps in this pathway take place within cells of the retinal pigment epithelium (RPE) adjacent to the photoreceptors. The key step in this pathway is all-trans to 11-cis re-isomerization of the retinoid, which is catalyzed by an enzyme activity called isomerohydrolase (IMH). IMH has been shown to use fatty acyl esters of retinol as a substrate (1, 2), harnessing the energy of ester hydrolysis [⌬G ϭ Ϫ5 kcal/mol (3)] for the endothermic conversion of all-trans-retinol (atROL) to 11-cis-retinol (11cROL) (ϩ4.1 kcal/mol, Ref. 4). IMH has never been purified or cloned. Leber's congenital amaurosis (LCA) is a severe and relatively common autosomal recessive disease that results in blindness at birth. LCA is frequently caused by mutations in the RP...
The findings of this study and the established safety profile of fenretinide in chronic dosing regimens warrant further study of fenretinide in the treatment of geographic atrophy.
Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to alltrans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical reisomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans-and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.Vision in vertebrates is mediated by two types of light-sensitive cells, rods and cones. Rods are specialized for vision in dim light, while cones provide high-resolution color vision in bright light. Despite the preponderance of rods in the human retina (∼95%), cones are more important for vision in civilized mankind. With the advent of artificial lighting, people spend most of their waking time under conditions where the rod response is saturated and vision is mediated entirely by cones.The first event in light perception is absorption of a photon by an opsin pigment molecule in the outer segment of a rod or cone. This induces 11-cis to all-trans isomerization of the retinaldehyde chromophore, which activates the opsin pigment and stimulates the visual transduction cascade (1). After a brief period, the photopigment decays to yield apoopsin and free all-trans-retinaldehyde (atRAL). 1 Before light sensitivity can be restored, the atRAL must be re-isomerized to 11-cis-retinaldehyde (11cRAL), which recombines with apoopsin to form a new rhodopsin or cone-opsin pigment molecule. The process of 11cRAL regeneration is called the visual cycle (Figure 1). This multistep pathway occurs within the retinal pigment † This work is supported by grants from the National Eye Institute (EY-11713) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript epithelium (RPE), a single layer of cells adjacent to the photoreceptors. The enzyme that catalyzes the critical all-trans to 11-cis isomerization step uses fatty-acyl esters of all-transretinol as the substrate (2-4). These all-trans-retinyl esters (atREs) are generated within the RPE by lecithin retinol acyl transferase (LRAT), which transfers a fatty acid from the sn-1 position of phosphati...
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