Publications

• Purified RPE65 shows isomerohydrolase activity after reassociation with a phospholipid membrane.

  Nikolaeva O., Takahashi Y., Moiseyev G., Ma J.X.

  Generation of 11-cis-retinol from all-trans-retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light. Recent findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the react ... >> More

  Generation of 11-cis-retinol from all-trans-retinyl ester in the retinal pigment epithelium is a critical step in the visual cycle and is essential for perception of light. Recent findings from cell culture models suggest that protein RPE65 is the retinoid isomerohydrolase that catalyzes the reaction. However, previous attempts to detect the enzymatic activity of purified RPE65 were unsuccessful, and thus its enzymatic function remains controversial. Here, we developed a novel liposome-based assay for isomerohydrolase activity. The results showed that purified recombinant chicken RPE65 had a high affinity for all-trans-retinyl palmitate-containing liposomes and demonstrated a robust isomerohydrolase activity. Furthermore, we found that all-trans-retinyl ester must be incorporated into the phospholipid membrane to serve as a substrate for isomerohydrolase. This assay system using purified RPE65 enabled us to measure kinetic parameters for the enzymatic reaction catalyzed by RPE65. These results provide conclusive evidence that RPE65 is the isomerohydrolase of the visual cycle. << Less

  FEBS J. 276:3020-3030(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• RPE65 is the isomerohydrolase in the retinoid visual cycle.

  Moiseyev G., Chen Y., Takahashi Y., Wu B.X., Ma J.X.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 102:12413-12418(2005) [PubMed] [EuropePMC]

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

  Kiser P.D., Golczak M., Lodowski D.T., Chance M.R., Palczewski K.

  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-ret ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 106:17325-17330(2009) [PubMed] [EuropePMC]

• Importance of membrane structural integrity for RPE65 retinoid isomerization activity.

  Golczak M., Kiser P.D., Lodowski D.T., Maeda A., Palczewski K.

  Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning dom ... >> More

  Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment. << Less

  J. Biol. Chem. 285:9667-9682(2010) [PubMed] [EuropePMC]

• Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium.

  Jin M., Li S., Moghrabi W.N., Sun H., Travis G.H.

  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 ... >> More

  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. << Less

  Cell 122:449-459(2005) [PubMed] [EuropePMC]

• An alternative isomerohydrolase in the retinal Muller cells of a cone-dominant species.

  Takahashi Y., Moiseyev G., Chen Y., Nikolaeva O., Ma J.X.

  Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cyc ... >> More

  Cone photoreceptors have faster light responses than rods and a higher demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) that converts all-trans retinyl ester to 11-cis retinol, a key step in the visual cycle for regenerating 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Müller cells, to meet the high demand for the chromophore by cones. In the present study, we describe the identification and characterization of a novel isomerohydrolase, RPE65c, from the cone-dominant zebrafish retina. RPE65c shares 78% amino acid sequence identity with RPE-specific zebrafish RPE65a (orthologue of human RPE65) and retains all of the known key residues for the enzymatic activity of RPE65. Similar to the other RPE-specific RPE65, RPE65c was present in both the membrane and cytosolic fractions, used all-trans retinyl ester as its substrate and required iron for its enzymatic activity. However, immunohistochemistry detected RPE65c in the inner retina, including Müller cells, but not in the RPE. Furthermore, double-immunostaining of dissociated retinal cells using antibodies for RPE65c and glutamine synthetase (a Müller cell marker), showed that RPE65c co-localized with the Müller cell marker. These results suggest that RPE65c is the alternative isomerohydrolase in the intra-retinal visual cycle, providing 11cRAL to cone photoreceptors in cone-dominant species. Identification of an alternative visual cycle will contribute to the understanding of the functional differences of rod and cone photoreceptors. << Less

  FEBS J. 278:2913-2926(2011) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase.

  Li S., Lee J., Zhou Y., Gordon W.C., Hill J.M., Bazan N.G., Miner J.H., Jin M.

  Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate an ... >> More

  Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children. Despite such importance, the mechanism that regulates RPE65 function remains unclear. Through unbiased expression screening of a bovine retinal pigment epithelium (RPE) cDNA library, we have identified elongation of very long-chain fatty acids-like 1 (ELOVL1) and fatty acid transport protein 4 (FATP4), which each have very long-chain fatty acid acyl-CoA synthetase (VLCFA-ACS) activity, as negative regulators of RPE65. We found that the VLCFA derivative lignoceroyl (C24:0)-CoA inhibited synthesis of 11cROL, whereas palmitoyl (C16:0)-CoA promoted synthesis of 11cROL. We further found that competition of FATP4 with RPE65 for the substrate of RPE65 was also involved in the mechanisms by which FATP4 inhibits synthesis of 11cROL. FATP4 was predominantly expressed in RPE, and the FATP4-deficient RPE showed significantly higher isomerase activity. Consistent with these results, the regeneration rate of 11-cis-retinaldehyde and the recovery rate for rod light sensitivity were faster in FATP4-deficient mice than wild-type mice. Moreover, FATP4-deficient mice displayed increased accumulation of the cytotoxic all-trans retinaldehyde and hypersusceptibility to light-induced photoreceptor degeneration. Our findings demonstrate that ELOVL1, FATP4, and their products comprise the regulatory elements of RPE65 and play important roles in protecting photoreceptors from degeneration induced by light damage. << Less

  J. Neurosci. 33:3178-3189(2013) [PubMed] [EuropePMC]