The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.
The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover blindness therapies. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatiydylcholine containing very long chain polyunsaturated fatty acids and severely-attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation, and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.
RPE). Interphotoreceptor retinoid-binding protein (IRBP) is present in the extracellular space between photoreceptors and the RPE. IRBP is known to bind visual retinoids. Previous studies on irbpϪ/Ϫ mice suggested that IRBP plays an insignificant role in opsin-pigment regeneration. However, the mice in these studies were uncontrolled for a severe mutation in the rpe65 gene. Rpe65 catalyzes the rate-limiting step in the visual cycle. Here, we examined the phenotype in irbp Ϫ/Ϫ mice homozygous for the wild-type (Leu450) rpe65 gene. We show that lack of IRBP causes delayed transfer of newly synthesized chromophore from RPE to photoreceptors. Removal of bleached chromophore from photoreceptors is also delayed in irbp Ϫ/Ϫ retinas after light exposure. It was previously shown that rods degenerate in irbp Ϫ/Ϫ mice. Here, we show that cones and rods degenerate at similar rates. However, cones are more affected functionally and show greater reductions in outer segment length than rods in irbp Ϫ/Ϫ mice. The disproportionate reductions in cone function and outer-segment length appear to result from mistrafficking of cone opsins due to impaired delivery of retinaldehyde chromophore, which functions as a chaperone for cone opsins but not rhodopsin.
Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-␣ (TNF-␣) in the interphotoreceptor matrix and retinas of Irbp Ϫ / Ϫ mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-␣ receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp Ϫ / Ϫ retina, and was colocalized with peanut agglutinin to the Irbp Ϫ / Ϫ cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp Ϫ / Ϫ retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp Ϫ / Ϫ retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp Ϫ / Ϫ retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp Ϫ / Ϫ mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp Ϫ / Ϫ mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.
Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of itsFurther, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.Vertebrate retinas contain light sensitive proteins called opsins, which are members of the G protein-coupled receptor superfamily. The light-absorbing chromophore in most vertebrate opsins is 11-cis-retinaldehyde (11-cis-RAL).2 Capture of a photon by an opsin pigment induces 11-cis to all-trans isomerization of the retinaldehyde chromophore. After a brief period of activation, the bleached pigment dissociates to yield apoopsin and free all-trans-retinaldehyde (all-trans-RAL). Restoration of light sensitivity to the apo-opsin requires chemical regeneration of 11-cis-RAL via an enzyme pathway called the visual cycle (Fig. 1). Most steps in this pathway take place within cells of the retinal pigment epithelium (RPE), which directly overlay the photoreceptors. Following its release by photoreceptors, all-trans-retinol (all-trans-ROL) is taken up by RPE cells and esterified to a fatty acid from phosphatidylcholine in internal membranes by lecithin:retinol acyl transferase (LRAT) (1). The retinoid isomerase uses an all-trans-retinyl ester (alltrans-RE), such as all-trans-retinyl palmitate (all-trans-RP) as substrate (2-4), harnessing the energy of ester hydrolysis to drive the endothermic (⌬G ϭ ϩ4.1 kcal/mol) isomerization reaction (5). The final catalytic step in the visual cycle involves oxidizing the 11-cis-retinol (11-cis-ROL) product of the isomerase to 11-cis-RAL (6, 7). Newly synthesized 11-cis-RAL is released by the RPE and taken up by photoreceptors where it combines with apo-opsin to form a new light sensitive pigment molecule. The retinoid isomerase was recently identified as an abundant 63-kDa protein in RPE cells called . Rpe65 is homologous to -carotene 15,15Ј-oxygenase in mammals (11), and apo-carotene oxygenase in cyanobacteria (12). By x-ray diffraction analysis, apo-carotene oxygenase is a seven-bladed -propeller (12). A similar structure is predicted for Rpe65 (13). Rpe65 associates with membranes, although it contains no predicted transmembrane segments (14). This association was shown to result from S-palmitoylation of residues Cys 231 , Cys 329 , and Cys 330 (15), which are on or near the protein surface (13). The acyl-transferase responsible for palmitoylation of Rpe65 is thought to be LRAT (15). A "palmitoylation switch" mechanism for the regulation of isomeras...
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