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Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms. [ABSTRACT FROM AUTHOR]

RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA. Here we show that R91W RPE65 undergoes ubiquitination-dependent proteasomal degradation in the knock-in mouse RPE due to misfolding. The 26S proteasome non-ATPase regulatory subunit 13 mediated degradation specifically of misfolded R91W RPE65. The mutation disrupted membrane-association and colocalization of RPE65 with lecithin:retinol acyltransferase (LRAT) that provides the hydrophobic substrate for RPE65. Systemic administration of sodium 4-phenylbutyrate (PBA), a chemical chaperone, increased protein stability, enzymatic activity, membrane-association, and colocalization of R91W RPE65 with LRAT. This rescue effect increased synthesis of 11-cis-retinal and 9-cis-retinal, a functional iso-chromophore of the visual pigments, led to alleviation of S-opsin mislocalization and cone degeneration in the knock-in mice. Importantly, PBA-treatment also improved conemediated vision in the mutant mice. These results indicate that PBA, a U.S. Food and Drug Administration-approved safe oral medication, may provide a noninvasive therapeutic intervention that delays daylight vision loss in patients with RPE65 mutations. [ABSTRACT FROM AUTHOR]

RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65. [ABSTRACT FROM AUTHOR]

Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fluorescence spectra of light- and dark-adapted bR and ASR protein samples reported. [•] The dark- and light-adapted ASR fluorescence spectra are fully characterized for the first time. [•] The pure retinal isomer fluorescence spectra, and quantum yields are deduced. [•] The average radiative lifetimes of the retinal isomers are found to be isomer-dependent. [•] Observation related to configuration-dependent transition dipole moment. [ABSTRACT FROM AUTHOR]

A large body of evidence for G-protein-coupled receptor (GPCR) oligomerization has accumulated over the past 2 decades. The smallest of these oligomers in vivo most likely is a dimer that buries 1000-Å2 intramolecular surfaces and on stimulation forms a complex with heterotrimeric G protein in 2:1 stoichiometry. However, it is unclear whether each of the monomers adopts the same or a different conformation and function after activation of this dimer. With bovine rhodopsin (Rho) and its cognate bovine G-protein transducin (Gt) as a model system, we used the retinoid chromophores 11-cis-retinal and 9-cis-retinal to monitor each monomer of the dimeric GPCR within a stable complex with nucleotide-free Gt. We found that only 50% of Rho* in the Rho*-Gt complex is trapped in a Meta II conformation, while 50% evolves toward an opsin conformation and can be regenerated with 9-cis-retinal. We also found that all-trans-retinal can regenerate chromophore-depleted Rho*e complexed with Gt and FAK*TSA peptide containing Lys296 with the attached all-trans retinoid (m/z of 934.5[MH]+) was identified by mass spectrometry. Thus, our study shows that each of the monomers contributes unequally to the pentameric (2:1:1:1) complex of Rho dimer and Gt heterotrimer, validating the oligomeric structure of the complex and the asymmetry of the GPCR dimer, and revealing its structural/functional signature. This study provides a clear functional distinction between monomers of family A GPCRs in their oligomeric form. [ABSTRACT FROM AUTHOR]

Abstract: The isolation of the retinal isomers from all-trans-retinal was performed by flash countercurrent chromatography. In each separation, isomerization reaction solution of 200mg all-trans-retinal could be loaded on a 1200mL of high-speed countercurrent chromatographic column with 5mm bore, eluted by a mobile phase flow rate of 25mL/min, resulting in 63mg of 11-cis-retinal, 24mg of 13-cis-retinal and 26mg of 9-cis-retinal with purities more than 95%. n-Hexane–acetonitrile (3:1) was used as the solvent system which possesses the advantages of simplicity, re-use of the solvent and multiple injections. This method could be used to prepare 13-cis-retinal, 11-cis-retinal and 9-cis-retinal for the photoisomerization investigation, such as the effect of 11-cis-retinal in the visual system. [Copyright &y& Elsevier]

Photoinduced biological processes occur via one-photon absorption in natural light, which is weak, continuous wave, and incoherent, but are often studied in the laboratory using pulsed coherent light. Here, we compare the response of a molecule to these two very different sources within a quantized radiation field picture. The latter is shown to induce coherent time evolution in the molecule, whereas the former does not. As a result, the coherent time dependence observed in the laboratory experiments will not be relevant to the natural biological process. Emphasis is placed on resolving confusions regarding this issue that are shown to arise from aspects of quantum measurement and from a lack of appreciation of the proper description of the absorbed photon. [ABSTRACT FROM AUTHOR]

Background: Best recognized for its role in manipulating host reproduction, the parasitic gram-negative Wolbachia pipientis is known to colonize a wide range of invertebrates. The endosymbiotic bacterium has recently been shown to cause a life-shortening effect as well as inhibiting replication of arboviruses in Aades aegypti; although the molecular mechanisms behind these effects are largely unknown. MicroRNAs (miRNAs) have been determined to have a wide range of roles in regulating gene expression in eukaryotes. A recent study showed that several A. aegypti mosquito miRNAs are differentially expressed when infected with Wolbachia. Methodology/Principal Findings: Based on the prior knowledge that one of these miRNAs, aae-miR-12, is differentially expressed in mosquitoes infected with Wolbachia, we aimed to determine any significance of this mediation. We also set out to characterize the target genes of this miRNA in the A. aegpyti genome. Bioinformatic approaches predicted a list of potential target genes and subsequent functional analyses confirmed that two of these, DNA replication licensing (MCM6) and monocarboxylate transporter (MCT1), are under the regulative control of aae-miR-12. We also demonstrated that aae-miR-12 is critical in the persistence of Wolbachia in the host cell. Conclusions/Significance: Our study has identified two target genes of aae-miR-12, a differentially expressed mosquito miRNA in Wolbachia-infected cells, and determined that the miRNA affects Wolbachia density in the host cells. [ABSTRACT FROM AUTHOR]

The isomerization ofall-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 eolocalize 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. [ABSTRACT FROM AUTHOR]

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 1 1-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-Å 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. [ABSTRACT FROM AUTHOR]

In animals, successful production of the visual chromophore (11- cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision. [ABSTRACT FROM AUTHOR]

In vertebrate retinal photoreceptors, the absorption of light by rhodopsin leads to photoisomerization of 11-cis-retinal to its all-trans isomer. To sustain vision, a metabolic system evolved that recycles all-trans-retinal back to 11-cis-retinal. The importance of this visual (retinoid) cycle is underscored by the fact that mutations in genes encoding visual cycle components induce a wide spectrum of diseases characterized by abnormal levels of specific retinoid cycle intermediates. In addition, intense illumination can produce retinoid cycle by-products that are toxic to the retina. Thus, inhibition of the retinoid cycle has therapeutic potential in physiological and pathological states. Four classes of inhibitors that include retinoid and nonretinoid compounds have been identified. We investigated the modes of action of these inhibitors by using purified visual cycle components and in vivo systems. We report that retinylamine was the most potent and specific inhibitor of the retinoid cycle among the tested compounds and that it targets the retinoid isomerase, RPE65. Hydrophobic primary amines like farnesylamine also showed inhibitory potency but a short duration of action, probably due to rapid metabolism. These compounds also are reactive nucleophiles with potentially high cellular toxicity. We also evaluated the role of a specific protein-mediated mechanism on retinoid cycle inhibitor uptake by the eye. Our results show that retinylamine is transported to and taken up by the eye by retinol-binding protein-independent and retinoic acid-responsive gene product 6-independent mechanisms. Finally, we provide evidence for a crucial role of lecithin: retinol acyltransferase activity in mediating tissue specific absorption and long lasting therapeutic effects of retinoid-based visual cycle inhibitors. [ABSTRACT FROM AUTHOR]

The visual (retinoid) cycle, the enzymatic pathway that regenerates chromophore after light absorption, is located primarily in the retinal pigment epithelium (RPE) and is essential for rod photoreceptor survival. Whether this pathway also is essential for cone photoreceptor survival is unknown, and there are no data from man or monkey to address this question. The visual cycle is naturally disrupted in humans with Leber congenital amaurosis (LCA), which is caused by mutations in RPE65, the gene that encodes the retinoid isomerase. We investigated such patients over a wide age range (3-52 years) for effects on the cone-rich human fovea. In vivo microscopy of the fovea showed that, even at the youngest ages, patients with RPE65-LCA exhibited cone photoreceptor loss. This loss was incomplete, however, and residual cone photoreceptor structure and function persisted for decades. Basic questions about localization of RPE65 and isomerase activity in the primate eye were addressed by examining normal macaque. RPE65 was definitively localized by immunocytochemistry to the central RPE and, by immunoblotting, appeared to concentrate in the central retina. The central retinal RPE layer also showed a 4-fold higher retinoid isomerase activity than more peripheral RPE. Early cone photoreceptor losses in RPE65-LCA suggest that robust RPE65-based visual chromophore production is important for cones; the residual retained cone structure and function support the speculation that alternative pathways are critical for cone photoreceptor survival. [ABSTRACT FROM AUTHOR]

Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys231, Cys329, and Cys330, and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat-/- mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat-/- mice. Substitution of Cys231, Cys329, and Cys330 with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, 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. [ABSTRACT FROM AUTHOR]

RPE65 is the retinal isomerase essential for conversion of all-trans-retinyl ester to 11-cis-retinol in the visual cycle. Leber's congenital amaurosis (LCA), an autosomal recessive form of RP resulting in blindness, is commonly caused by mutations in the Rpe65 gene. Whereas the molecular mechanisms by which these mutations contribute to retinal disease remain largely unresolved, affected patients show marked RPE damage and photoreceptor degeneration. We evaluated gene expression in Rpe65-/- mouse model of LCA before and at the onset of photoreceptor cell death in 2, 4, and 6 month old animals. Microarray analysis demonstrates altered expression of genes involved in phototransduction, apoptosis regulation, cytoskeleton organization, and extracellular matrix (ECM) constituents. Cone-specific phototransduction genes are strongly decreased, reflecting early loss of cones. In addition, remaining rods show modified expression of genes encoding components of the cytoskeleton and ECM. This may affect rod physiology and interaction with the adjacent RPE and lead to loss of survival signals, as reflected by the alteration of apoptosis-related genes Together, these results suggest that RPE65 defect triggers an overall remodeling of the neurosensitive retina that may, in turn, disrupt photoreceptor homeostasis and induce apoptosis signaling cascade toward retinal cell death. [ABSTRACT FROM AUTHOR]

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 all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization 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. [ABSTRACT FROM AUTHOR]

Summary: 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. [Copyright &y& Elsevier]

Background: The vertebrate opsins are proteins which utilise a retinaldehyde chromophore in their photosensory or photoisomerase roles in the visual/irradiance detection cycle. The majority of the opsins, such as rod and cone opsins, have a very highly conserved gene structure suggesting a common lineage. Exceptions to this are RGR-opsin and melanopsin, whose genes have very different intron insertion positions. The gene structure of another opsin, peropsin (retinal pigment epithelium-derived rhodopsin homologue, RRH) is unknown. Results: By in silico analysis of the GenBank database we have determined that the human RRH comprises 7 exons spanning approximately 16.5 kb and is localised to chromosome 4q25 in the following gene sequence: cen-EGF-RRH-IF-qter -- a position that excludes this gene as a candidate for the RP29 autosomal recessive retinitis pigmentosa locus. A comparison of opsin gene structures reveals that RRH and RGR share two common intron (introns 1 and 4) insertion positions which may reflect a shared ancestral gene. Conclusion: The opsins comprise a diverse group of genes which appear to have arisen from three different lineages. These lineages comprise the "classical opsin superfamily" which includes the rod and cone opsins, pinopsin, VA-opsin, parapinopsin and encephalopsin; the RRH and RGR group; and the melanopsin line. A common lineage for RRH and RGR, together with their sites of expression in the RPE, indicates that peropsin may act as a retinal isomerase. [ABSTRACT FROM AUTHOR]