Article

Rpe65 Is the Retinoid Isomerase in Bovine Retinal Pigment Epithelium

Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
Cell (Impact Factor: 32.24). 09/2005; 122(3):449-59. DOI: 10.1016/j.cell.2005.06.042
Source: PubMed

ABSTRACT

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.

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Available from: Gabriel H. Travis, Jun 30, 2014
    • "Importantly, the RPE takes an active part in the so-called visual cycle, as it expresses the enzymes required for re-isomerization of all-trans retinal to 11-cis retinal and for its transport back to photoreceptors to regenerate visual pigments and complete the visual cycle [e.g. LRAT, RPE65, CRALBP (RLBP1), IRBP (RBP3)] (Batten et al., 2004; Jin et al., 2005; Travis et al., 2007). All-trans retinol is also supplied to the RPE by the choroidal vasculature, entering the RPE in a receptor-mediated process involving recognition of a serum retinolbinding protein/transthyretin (RBP-TTR) complex (Thompson and Gal, 2003). "
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    ABSTRACT: Vision incapacitation and blindness associated with incurable retinal degeneration affect millions of people worldwide. In this study, 0.25×10(6) human bone marrow stem cells (hBM-MSCs) were transplanted epiretinally in the right eye of Royal College Surgeons (RCS) rats at the age of 28days. Epiretinally transplanted cells were identified as a thin layer of cells along vitreous cavity, in close proximity to the retina or attached to the lens capsule, up to 6weeks following transplantation. Epiretinal transplantation delayed photoreceptor degeneration and rescued retinal function up to 20weeks following cell transplantation. Visual functions remained close to normal levels in epiretinal transplantation rats. No inflammation or any other adverse effects were observed in transplanted eyes. Our findings suggest that transplantation of hBM-MSCs as a thin epiretinal layer is effective for treatment of retinal degeneration in RCS rats, and that transplanting the cells in close proximity to the retina enhances hBM-MSC therapeutic effect compared with intravitreal injection. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Aug 2015 · Stem Cell Research
    • "Importantly, the RPE takes an active part in the so-called visual cycle, as it expresses the enzymes required for re-isomerization of all-trans retinal to 11-cis retinal and for its transport back to photoreceptors to regenerate visual pigments and complete the visual cycle [e.g. LRAT, RPE65, CRALBP (RLBP1), IRBP (RBP3)] (Batten et al., 2004; Jin et al., 2005; Travis et al., 2007). All-trans retinol is also supplied to the RPE by the choroidal vasculature, entering the RPE in a receptor-mediated process involving recognition of a serum retinolbinding protein/transthyretin (RBP-TTR) complex (Thompson and Gal, 2003). "
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    ABSTRACT: Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.
    No preview · Article · Aug 2015 · Journal of Cell Science
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    • "The formation of a thioester intermediate resulting from the addition of an acyl chain on Cys161 has clearly been demonstrated by mass spectrometry after incubation of tLRAT with phosphatidylcholine substrates [14]. This esterified form of retinol can thereby be accumulated in microsomes for storage, or hydrolyzed and isomerized by RPE65 to form 11- cis-retinol [16] [17] which is then further metabolized to produce the chromophore of rhodopsin (for a review, see [7,18–22]). "
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    ABSTRACT: Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in details. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100°C. The results of this study highly improved our understanding of this enzyme.
    Full-text · Article · Jun 2014 · Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics
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