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RPE65 is the isomerohydrolase in the retinoid visual cycle.

ABSTRACT: RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol/min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.

SUBMITTER: Moiseyev G

PROVIDER: S-EPMC1194921 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Crystal structure of native RPE65, the retinoid isomerase of the visual cycle.

Project description:Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-A resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

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FATP1 inhibits 11-cis retinol formation via interaction with the visual cycle retinoid isomerase RPE65 and lecithin:retinol acyltransferase.

Project description:The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.

| S-EPMC2881799 | biostudies-literature

The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.

Project description:Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B. In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place. We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle. We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells. This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light. RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels. Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity. Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction. Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.

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Aromatic lipophilic spin traps effectively inhibit RPE65 isomerohydrolase activity.

Project description:We previously showed that RPE65 does not specifically produce 11-cis-retinol only but also 13-cis-retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-α-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO), and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro.

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Chemistry of the retinoid (visual) cycle.

Project description: Not available

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Identification of key residues determining isomerohydrolase activity of human RPE65.

Project description:RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met(1)-Arg(33)) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.

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Focus on Molecules: RPE65, the visual cycle retinol isomerase.

Project description: Not available

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Inner retinal photoreception independent of the visual retinoid cycle.

Project description:Mice lacking the visual cycle enzymes RPE65 or lecithin-retinol acyl transferase (Lrat) have pupillary light responses (PLR) that are less sensitive than those of mice with outer retinal degeneration (rd/rd or rdta). Inner retinal photoresponses are mediated by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs), suggesting that the melanopsin-dependent photocycle utilizes RPE65 and Lrat. To test this hypothesis, we generated rpe65(-/-); rdta and lrat(-/-); rd/rd mutant mice. Unexpectedly, both rpe65(-/-); rdta and lrat(-/-); rd/rd mice demonstrate paradoxically increased PLR photosensitivity compared with mice mutant in visual cycle enzymes alone. Acute pharmacologic inhibition of the visual cycle of melanopsin-deficient mice with all-trans-retinylamine results in a near-total loss of PLR sensitivity, whereas treatment of rd/rd mice has no effect, demonstrating that the inner retina does not require the visual cycle. Treatment of rpe65(-/-); rdta with 9-cis-retinal partially restores PLR sensitivity. Photic sensitivity in P8 rpe65(-/-) and lrat(-/-) ipRGCs is intact as measured by ex vivo multielectrode array recording. These results demonstrate that the melanopsin-dependent ipRGC photocycle is independent of the visual retinoid cycle.

| S-EPMC1502474 | biostudies-literature

A novel RPE65 inhibitor CU239 suppresses visual cycle and prevents retinal degeneration.

Project description:The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 μM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.

| S-EPMC5963526 | biostudies-literature

RPE65: role in the visual cycle, human retinal disease, and gene therapy.

Project description:RPE65 is an isomerohydrolase expressed in retinal pigment epithelium. It is critical for the regeneration of the visual pigment necessary for both rod and cone-mediated vision. Mutations in human RPE65 cause Leber's congenital amaurosis and other forms of autosomal recessive retinitis pigmentosa which are associated with early-onset blindness. Several RPE65 animal models including two different mouse models and a naturally occurring canine model have been thoroughly characterized to determine the mechanisms that underlie RPE65 associated retinal dystrophies. More recently, substantial effort has gone into designing gene therapies for these diseases. Based on several encouraging reports from animal models, at least three clinical trials are currently underway for the treatment of LCA using modified AAV vectors carrying the RPE65 cDNA and have reported positive preliminary results.

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