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Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice
Journal of Biological Chemistry, 2015Co-Authors: Bhubanananda Sahu, Krzysztof Palczewski, Wenyu Sun, Lindsay Perusek, Vipulkumar Parmar, Michael D. Griswold, Akiko MaedaAbstract:Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-Retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-Retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third Retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5(-/-)Rdh11(-/-) mice as compared with Rdh5(-/-) mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other Retinol dehydrogenases was observed in both Rdh5(-/-) and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.
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Expansion of First-in-Class Drug Candidates That Sequester Toxic All-Trans-Retinal and Prevent Light-Induced Retinal Degeneration
Molecular pharmacology, 2014Co-Authors: Jianye Zhang, Krzysztof Palczewski, Zhiqian Dong, Sreenivasa R. Mundla, William L. Seibel, Ruben Papoian, Marcin GolczakAbstract:All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:Retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-Retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-Retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.
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retinyl ester storage particles retinosomes from the retinal pigmented epithelium resemble lipid droplets in other tissues
Journal of Biological Chemistry, 2011Co-Authors: Tivadar Orban, Grazyna Palczewska, Krzysztof PalczewskiAbstract:Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:Retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-Retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored.
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evaluation of 9 cis retinyl acetate therapy in rpe65 mice
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Tadao Maeda, Krzysztof Palczewski, Akiko Maeda, Gemma Casadesus, Philippe MargaronAbstract:Visual perception results from the biological conversion of light energy to electrical signaling by retinal photoreceptors in the eye, a process called phototransduction. Visual pigments, consisting of the chromophore 11-cis-retinal bound to apoprotein G protein-coupled receptor opsins,1 initiate this process on the absorption of a photon that triggers photoisomerization of the chromophore into its trans form.1,2 The isomerized chromophore, all-trans-retinal, then is reduced to all-trans-Retinol, transported to the retinal pigment epithelium (RPE), and converted to fatty acid all-trans-retinyl esters by lecithin/Retinol acyltransferase (LRAT). Regeneration of 11-cis-retinal completes this retinoid (visual) cycle and is critical for maintaining vision.3 Defects in 11-cis-retinal production are associated with a number of inherited degenerative retinopathies.4 Two examples are Leber congenital amaurosis (LCA), a childhood-onset retinal disease causing severe visual impairment, and retinitis pigmentosa (RP), another retinopathy with a more variable age of onset. At or soon after birth, LCA patients characteristically exhibit severe visual impairment exhibited by wandering nystagmus, amaurotic pupils, a pigmentary retinopathy with loss of cone and rod sensitivity, absent or greatly attenuated electroretinographic (ERG) responses, and an approximately 100-fold decrease in cone flicker amplitudes.5–7 RPE65, a 65-kDa protein specific to and abundant in the RPE8 that catalyzes the isomerization of fatty acid all-trans-retinyl esters to 11-cis-Retinol, has been recently described as the retinoid isomerase involved in the regeneration of 11-cis-retinal.9–11 Mutations in the RPE65 gene account for up to 16% of LCA cases and 2% of autosomal recessive RP cases.4,12–15 Spontaneous or engineered deletions of Rpe65 in mice and dogs result in 11-cis-retinal deficiency, an early-onset and slowly progressive retinal degeneration with dramatically reduced ERG responses and typical LCA pathology16–19 accompanied by an accumulation of fatty acid all-trans-retinyl esters in the RPE.16,20 LCA is incurable, but several possible therapies are being investigated. RPE65 gene augmentation therapy and retinal prostheses have shown preliminary encouraging signs of visual rescue in early-stage clinical evaluations.21–23 Recently, visual chromophore replacement therapy with 9-cis-retinal has been proposed as a novel pharmacologic approach to bypass the defective retinoid cycle.24–27 9-cis-Retinal binds to opsin to form the rod cell pigment, iso-rhodopsin, which initiates phototransduction similar to that of rhodopsin.1 Oral administration of 9-cis-retinal or its precursors have regenerated opsin as iso-rhodopsin in the eyes, improved retinal function as assessed by ERG responses, and ameliorated the pupillary light reflex in Rpe65 and Lrat knockout mice, two genetic models of LCA.24–27 These observations have led to the development of synthetic 9-cis-retinoids as orally administered, prodrug forms of 9-cis-retinal for the treatment of various forms of inherited retinal degeneration caused by defects in the retinoid cycle. Here we report pharmacokinetic and pharmacodynamic effects of the prodrug 9-cis-R-Ac that is converted to another prodrug in the liver (i.e., mostly to 9-cis-retinyl palmitate) in the Rpe65−/− mouse model. We describe the selection of soybean oil as an appropriate vehicle for administering the 9-cis-R-Ac prodrug by gastric gavage, based on postabsorptive levels of its pharmacologically active metabolites in plasma. We assessed 9-cis-R-Ac efficacy with the use of electroretinography and vision-dependent behavioral tests in single, intermittent, and daily dosing studies that also included biochemical quantification of retinoids in the eye. Dose-dependent improvement of the level and duration of retinal function was observed in these knockout animals. Importantly, pharmacologic activity was sustained for sufficiently long periods after dosing to enable the formulation of a flexible, intermittent dosing schedule.
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Retinyl Ester Homeostasis in the Adipose Differentiation-related Protein-deficient Retina
The Journal of biological chemistry, 2008Co-Authors: Yoshikazu Imanishi, Tadao Maeda, Akiko Maeda, Wenyu Sun, Krzysztof PalczewskiAbstract:The retinal pigmented epithelium (RPE) plays an essential role in vision, including storing and converting retinyl esters of the visual chromophore, 11-cis-retinal. Retinyl ester storage structures (RESTs), specialized lipid droplets within the RPE, take up retinyl esters synthesized in the endoplasmic reticulum. Here we report studies of mice lacking exons 2 and 3 of the gene encoding adipose differentiation-related protein (Adfp), a structural component of RESTs. We found that dark adaptation was slower in AdfpΔ2-3/Δ2-3 than in Adfp+/+ mice and that AdfpΔ2-3/Δ2-3 mice had consistently delayed clearances of all-trans-retinal and all-trans-Retinol from rod photoreceptor cells. Two-photon microscopy revealed aberrant trafficking of all-trans-retinyl esters in the RPE of AdfpΔ2-3/Δ2-3 mice, a problem caused by abnormal maintenance of RESTs in the dark-adapted state. Retinyl ester accumulation was also reduced in AdfpΔ2-3/Δ2-3 as compared with Adfp+/+ mice. These observations suggest that Adfp plays a unique role in vision by maintaining proper storage and trafficking of retinoids within the eye.
Yiannis Koutalos - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein removes all-trans-Retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.
Journal of Biological Chemistry, 2017Co-Authors: Chunhe Chen, Leopold Adler, Patrice Goletz, Debra A. Thompson, Federico Gonzalez-fernandez, Yiannis KoutalosAbstract:: Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and Retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the Retinol dehydrogenase RDH8 to all-trans-Retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-Retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-Retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for Retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 μm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to Retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and Retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.
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Microfluorometric measurement of the formation of all-trans-Retinol in the outer segments of single isolated vertebrate photoreceptors.
Methods of Molecular Biology, 2010Co-Authors: Yiannis Koutalos, M. Carter CornwallAbstract:: The first step in the detection of light by vertebrate photoreceptors is the photoisomerization of the retinyl chromophore of their visual pigment from 11-cis to the all-trans configuration. This initial reaction leads not only to an activated form of the visual pigment, meta II, that initiates reactions of the visual transduction cascade but also to the photochemical destruction of the visual pigment. By a series of reactions termed the visual cycle, native visual pigment is regenerated. These coordinated reactions take place in the photoreceptors themselves as well as the adjacent pigment epithelium and Muller cells. The critical initial steps in the visual cycle are the release of all-trans-retinal from the photoactivated pigment and its reduction to all-trans-Retinol. The goal of this monograph is to describe methods of fluorescence imaging that allow the measurement of changes in the concentration of all-trans-Retinol as it is reduced from all-trans-retinal in isolated intact salamander and mouse photoreceptors. The kinetics of all-trans-Retinol formation depend on cellular factors that include the visual pigment and photoreceptor cell type, as well as the cytoarchitecture of outer segments. In general, all-trans-Retinol forms much faster in cone cells than in rods.
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Formation of all-trans Retinol after visual pigment bleaching in mouse photoreceptors.
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Chunhe Chen, Lorie R. Blakeley, Yiannis KoutalosAbstract:Vision is initiated by the absorption of light by the visual pigment present in the outer segments of the rod and cone photoreceptor cells in the retina. In both cell types, the first step in the detection of light is the photoisomerization of the retinyl chromophore of the visual pigment from 11-cis to all-trans.1,2 This isomerization of the chromophore bleaches the pigment, necessitating its regeneration with fresh 11-cis retinal. The production of 11-cis retinal includes the recycling of the all-trans chromophore of the bleached pigment through a series of reactions called the visual cycle.3–5 These reactions begin in the outer segment with the release of all-trans retinal from the photoactivated pigment and its reduction to all-trans Retinol by Retinol dehydrogenase in a reaction using NADPH.6 The all-trans Retinol is then transferred from outer segments to the adjacent retinal pigment epithelial cells,7 where it is esterified to form retinyl ester.8,9 The ester is converted to 11-cis Retinol,10–13 which is then oxidized to 11-cis retinal.14 Studies of cone-dominant ground squirrel and chicken retinas have provided evidence of the presence of an additional visual cycle used by cones.15–17 Continuous vision depends on the regeneration of the visual pigment and defects in the processing of the chromophore through the reactions of the visual cycle are responsible for a wide range of visual defects.3,18 Extensive studies using whole eyes have argued for the presence of two slow steps in the operation of the mouse visual cycle: the formation of all-trans Retinol and the isomerization of all-trans retinyl ester.19–21 Although the formation of all-trans Retinol has been characterized in detail in amphibian photoreceptors,22–24 these results cannot provide a quantitative insight into the operation of the mouse visual cycle because of critical species differences, such as body temperature. Therefore, we undertook the measurement of the kinetics of all-trans Retinol formation in mouse photoreceptors by HPLC of retinoid extracts and fluorescence imaging. In our results, all-trans Retinol formation was not the slowest step in the mouse visual cycle, which supports the notion that the isomerization of retinyl esters is the slowest step.20,21 Throughout the text, unqualified retinal and Retinol refer to the all-trans isomers.
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Formation Of All-Trans Retinol In Mouse Rod Photoreceptors
Biophysical Journal, 2009Co-Authors: Lorie R. Blakeley, Chunhe Chen, Yiannis KoutalosAbstract:Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans retinal and its reduction to all-trans Retinol. All-trans Retinol is then transported to the neighboring retinal pigment epithelial cells where it is used to remake 11-cis retinal. The reduction of all-trans retinal to all-trans Retinol is catalyzed by Retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans Retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans Retinol formation proceeds with a rate of ∼0.06 min−1, which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans retinal, as formation of all-trans Retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans Retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans Retinol formation on the level of metabolic activity.
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interphotoreceptor retinoid binding protein is the physiologically relevant carrier that removes Retinol from rod photoreceptor outer segments
Biochemistry, 2007Co-Authors: Qingqing Wu, Barbara N Wiggert, Lorie R. Blakeley, Carter M Cornwall, Yiannis KoutalosAbstract:The vertebrate cells responsible for vision are the rod and cone photoreceptors of the retina that convert incoming light to an electrical signal. This conversion takes place in the photoreceptor outer segments, which are full of membrane disks containing the visual pigment, and, in a physiologically important arrangement, are enveloped by the retinal pigment epithelium (RPE). The visual pigment is composed of a chromophore, 11-cis retinal, attached to an integral membrane protein, opsin. The detection of light begins with the absorption of incoming photons by the visual pigment. An absorbed photon isomerizes the chromophore moiety from 11-cis to all-trans bringing about a conformational change that initiates a cascade of reactions culminating in membrane potential change. The recovery of the cell from light involves the deactivation of the intermediates activated by light, and the reestablishment of membrane potential (1, 2). However, the isomerized chromophore, all-trans retinal, remains. For vision to be possible, it is essential that the visual pigment regenerate: that is, the all-trans retinal has to be removed, and fresh 11-cis retinal has to be provided to combine with opsin and reform the visual pigment. The reactions regenerating the pigment are known as the Visual Cycle (3–5). In the case of the rod photoreceptors, the cells responsible for vision at low light intensities, the Visual Cycle encompasses reactions in the outer segment and in the adjacent RPE cells. The first step in the Cycle is the release of all-trans retinal from photoactivated rhodopsin after hydrolysis of the Schiff base bond linking the chromophore to opsin. All-trans retinal is then reduced to all-trans Retinol in a reaction catalyzed by Retinol dehydrogenase (6, 7), requiring NADPH, and taking place on the cytoplasmic side of the outer segment disks. It is possible that all-trans retinal ends up inside the disks, bound via a Schiff base to phosphatidylethanolamine, in which case the phosphatidylethanolamine-all-trans-retinal compound is transported to the cytoplasmic side by the ABCR transporter (8–10) making all-trans retinal available for reduction. The all-trans Retinol formed in the rod outer segment is transported to the RPE, in a process that can be facilitated by the interphotoreceptor retinoid binding protein (IRBP; (11–13)). In the RPE, Retinol is converted by lecithin-Retinol acyltransferase to retinyl ester (LRAT; (14, 15)), which is isomerized to 11-cis Retinol (16, 17) by the RPE65 protein (18–21). 11-cis Retinol is then oxidized to 11-cis retinal, and transported back to photoreceptor outer segments where it associates with opsin to reform rhodopsin. Previous work (22–25) has established that all-trans Retinol can be monitored in the outer segments of living isolated rod and cone photoreceptors from its distinctive fluorescence. Here, we have taken advantage of two properties of frog rod photoreceptors to actually measure the amounts of all-trans Retinol produced with quantitative biochemical and physiological methods. One, in contrast to larval salamander photoreceptors that contain two types of chromophore (based on vitamins A1 and A2) and in widely varying ratios (24), frog rods contain a single, vitamin A1-based chromophore. Two, the metabolic supply of NADPH is not limiting for the formation of all-trans Retinol in the case of frog rods (23), allowing a simplified analysis and direct comparisons between biochemical and physiological data. This has further allowed us to properly characterize the removal of all-trans Retinol by different lipophilic carriers. In experiments with purified rod outer segment membranes, whole retinas, and living isolated rods, we have separately measured the different steps involved in all-trans Retinol formation and removal. On the basis of these measurements, we have calculated the predicted kinetics of Retinol formation and removal in rod outer segments, and found that they are in close agreement with those measured directly from isolated rod photoreceptors. We also characterized the effect of different concentrations of lipophilic carriers on the removal of all-trans Retinol, and established that for the physiological concentrations of carriers the rate of all-trans Retinol removal is determined by the IRBP concentration. Our results strongly support a specific interaction mediating the removal of all-trans Retinol by IRBP, perhaps through a receptor on the rod outer segment plasma membrane. Throughout the text, when not specifically designated as the 11-cis isomers, unqualified retinal and Retinol refer to the all-trans forms.
Robert R. Rando - One of the best experts on this subject based on the ideXlab platform.
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RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters.
Biochemistry, 2003Co-Authors: Deviprasad R. Gollapalli, Pranab Maiti, Robert R. RandoAbstract:RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K D = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K D = 14 nM, 11-cis-Retinol binds with a K D = 3.8 nM, and all-trans-Retinol (vitamin A) binds with a K D = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by birding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.
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All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle.
Biochemistry, 2003Co-Authors: Deviprasad R. Gollapalli, Robert R. RandoAbstract:The identification of the critical enzyme(s) that carries out the trans to cis isomerization producing 11-cis-Retinol during the operation of the visual cycle remains elusive. Confusion exists in the literature as to the exact nature of the isomerization substrate. At issue is whether it is an all-trans-retinyl ester or all-trans-Retinol (vitamin A). As both putative substrates interconvert rapidly in retinal pigment epithelial membranes, the choice of substrate can be ambiguous. The two enzymes that effect interconversion of all-trans-Retinol and all-trans-retinyl esters are lecithin Retinol acyl transferase (LRAT) and retinyl ester hydrolase (REH). The retinyl ester or all-trans-Retinol pools are radioactively labeled separately in the presence of inhibitors of LRAT and REH, effectively preventing their interconversion. Pulse-chase experiments unambiguously demonstrate that all-trans-retinyl esters, and not all-trans-Retinol, are the precursors of 11-cis-Retinol. When the all-trans-retinyl ester pool is radioactively labeled, the resulting 11-cis-Retinol is labeled with the same specific activity as the precursor ester. The converse is true with vitamin A. These data unambiguously establish all-trans-retinyl esters as the precursors of 11-cis-Retinol.
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molecular and biochemical characterization of lecithin Retinol acyltransferase
Journal of Biological Chemistry, 1999Co-Authors: Alberto Ruiz, Anette Winston, Bryant A Gilbert, Robert R. RandoAbstract:Abstract The enzyme responsible for conversion of all-trans-Retinol into retinyl esters, the lecithin Retinol acyltransferase (LRAT) has been characterized at the molecular level. The cDNA coding for this protein was cloned and its amino acid sequence deduced. LRAT is composed of a polypeptide of 230 amino acid residues with a calculated mass of 25.3 kDa. Tissue distribution analysis by Northern blot showed expression of a 5.0-kilobase transcript in the human retinal pigment epithelium as well as in other tissues that are known for their high LRAT activity and vitamin A processing. Affinity labeling experiments using specific compounds with high affinity for LRAT and monospecific polyclonal antibodies raised in rabbits against two peptide sequences for LRAT confirmed the molecular mass of LRAT as a 25-kDa protein. High performance liquid chromatography analysis of the reaction product formed by HEK-293 cells transfected with LRAT cDNA confirmed the ability of the transfected cells to convert [3H]all-trans-Retinol into authentic [3H]all-trans-retinyl palmitate as chemically determined.
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Lack of effect of RPE65 removal on the enzymatic processing of all‐trans‐Retinol into 11‐cis‐Retinol in vitro
FEBS Letters, 1998Co-Authors: Dong Won Choo, Eric Cheung, Robert R. RandoAbstract:RPE65 is a major membrane associated protein found in the vertebrate retinal pigment epithelium (RPE). Various studies have shown this protein to be essential for visual function, possibly at the level of the processing of retinoids. The pigment epithelium is the anatomical site in which the visual chromophore 11-cis retinal is generated. The two critical RPE enzymes in the isomerization pathway are lecithin Retinol acyl transferase (LRAT) and isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-Retinol. Both enzymes are membrane bound. It is shown here that RPE65 can be largely extracted (90–95%) from RPE membranes by 1 M KCl by itself, or with added detergent CHAPS. The almost quantitative extraction of RPE65 from RPE membranes has little or no effect on in vitro LRAT and isomerohydrolase activities in quantitative enzymatic assays using RPE membranes, suggesting that RPE65 may not have an important role to play in the enzymatic processing of all-trans-Retinol into 11-cis-Retinol in vitro.
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Lack of effect of RPE65 removal on the enzymatic processing of all-trans-Retinol into 11-cis-Retinol in vitro.
FEBS letters, 1998Co-Authors: Dong Won Choo, Eric Cheung, Robert R. RandoAbstract:RPE65 is a major membrane associated protein found in the vertebrate retinal pigment epithelium (RPE). Various studies have shown this protein to be essential for visual function, possibly at the level of the processing of retinoids. The pigment epithelium is the anatomical site in which the visual chromophore 11-cis retinal is generated. The two critical RPE enzymes in the isomerization pathway are lecithin Retinol acyl transferase (LRAT) and isomerohydrolase, which processes all-trans-retinyl esters into 11-cis-Retinol. Both enzymes are membrane bound. It is shown here that RPE65 can be largely extracted (90-95%) from RPE membranes by 1 M KCl by itself, or with added detergent CHAPS. The almost quantitative extraction of RPE65 from RPE membranes has little or no effect on in vitro LRAT and isomerohydrolase activities in quantitative enzymatic assays using RPE membranes, suggesting that RPE65 may not have an important role to play in the enzymatic processing of all-trans-Retinol into 11-cis-Retinol in vitro.
Sylvia B. Smith - One of the best experts on this subject based on the ideXlab platform.
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© Molecular Vision Analysis of esterification of retinoids in the retinal pigmented epithelium of the Mitf vit (vitiligo) mutant mouse
2013Co-Authors: Bill L. Evans, Sylvia B. SmithAbstract:Purpose: Mice homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene have a retinal degeneration characterized by slow loss of photoreceptor cells and compromised retinal pigment epithelial (RPE) structure and function. The levels of retinyl esters, which are essential for generation of 11-cis-retinaldehyde for the formation of rhodopsin, were reported previously to be elevated by 6 weeks postnatally in the RPE of vitiligo mutant mice. The purpose of the present study was to determine whether this elevation was due to increased activity of lecithin:Retinol acyl transferase (LRAT) the enzyme that converts all-trans-Retinol to retinyl esters. Methods: Retinoids extracted from the RPE and neural retina of mutant and normal mice ages 2, 4, 6 and 8 weeks were analyzed by reversed-phase HPLC. The esterification capacity of the RPE to convert 3 H-Retinol to 3 H retinyl ester was determined by HPLC in mutant and normal mice at 3 and 9 weeks. Results: Retinyl ester levels were elevated significantly in the mutant RPE as early as postnatal week 2 and were four-fold greater by 8 weeks. The esterification assay indicated no significant differences between mutants and controls at 3 weeks. At 9 weeks, the esterification activity of the mutant RPE was significantly reduced compared to controls rather than elevated. Conclusions: The data suggest that the accumulation of retinyl esters is not due to increased LRAT activity. Alternative explanations for the retinyl ester accumulation are discussed
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analysis of esterification of retinoids in the retinal pigmented epithelium of the mitf vit vitiligo mutant mouse
Molecular Vision, 1997Co-Authors: Bill L. Evans, Sylvia B. SmithAbstract:Purpose: Mice homozygous for the vitiligo mutation of the microphthalmia ( Mitf) gene have a retinal degeneration characterized by slow loss of photoreceptor cells and compromised retinal pigment epithelial (RPE) structure and function. The levels of retinyl esters, which are essential for generation of 11- cis-retinaldehyde for the formation of rhodopsin, were reported previously to be elevated by 6 weeks postnatally in the RPE of vitiligo mutant mice. The purpose of the present study was to determine whether this elevation was due to increased activity of lecithin:Retinol acyl transferase (LRAT) the enzyme that converts all-trans-Retinol to retinyl esters. Methods: Retinoids extracted from the RPE and neural retina of mutant and normal mice ages 2, 4, 6 and 8 weeks were analyzed by reversed-phase HPLC. The esterification capacity of the RPE to convert 3 H-Retinol to 3 H retinyl ester was determined by HPLC in mutant and normal mice at 3 and 9 weeks. Results: Retinyl ester levels were elevated significantly in the mutant RPE as early as postnatal week 2 and were four-fold greater by 8 weeks. The esterification assay indicated no significant differences between mutants and controls at 3 weeks. At 9 weeks, the esterification activity of the mutant RPE was significantly reduced compared to controls rather than elevated. Conclusions: The data suggest that the accumulation of retinyl esters is not due to increased LRAT activity. Alternative explanations for the retinyl ester accumulation are discussed.
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increase in retinyl palmitate concentration in eyes and livers and the concentration of interphotoreceptor retinoid binding protein in eyes of vitiligo mutant mice
Biochemical Journal, 1994Co-Authors: Sylvia B. Smith, Todd Duncan, Geetha Kutty, R K Kutty, Barbara WiggertAbstract:Retinyl esters play an important role in the visual cycle because they are involved in regeneration of 11-cis-retinal for use in rhodopsin formation. In the present study, retinyl ester concentrations were significantly elevated in eyes and livers of mice homozygous for the vitiligo mutation (mivit/mivit). Vitiligo mice demonstrate a slowly progressing retinal degeneration characterized by gradual loss of photoreceptor cells and rhodopsin as well as uneven pigmentation of the retinal pigment epithelium (RPE). Analysis of retinoids by h.p.l.c. indicated that the retinyl palmitate level was increased fivefold in eyes of affected mice at 10 weeks postnatally and was threefold higher at 22 weeks of age. Accumulation of retinyl palmitate occurred in the RPE rather than the neural retina. Furthermore, the concentration of all-trans-Retinol was elevated in the RPE of vitiligo mice. Levels of interphotoreceptor retinoid binding protein (IRBP) were increased in vitiligo mice between ages 4 and 14 weeks, but returned to normal by 16 weeks. Increased IRBP levels were not due to increased protein synthesis because IRBP mRNA levels did not differ significantly between control and affected animals. To examine possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Mean hepatic total vitamin A levels in affected mice were approximately 1.7 times higher than controls. Analysis of esterified and non-esterified retinoids in liver showed that the concentration of retinyl palmitate was elevated. Plasma Retinol levels were normal. This study provides the first evidence of altered systemic retinoid metabolism in vitiligo mice, which occurs, significantly, under normal dietary conditions.
Chunhe Chen - One of the best experts on this subject based on the ideXlab platform.
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Interphotoreceptor retinoid-binding protein removes all-trans-Retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.
Journal of Biological Chemistry, 2017Co-Authors: Chunhe Chen, Leopold Adler, Patrice Goletz, Debra A. Thompson, Federico Gonzalez-fernandez, Yiannis KoutalosAbstract:: Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and Retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the Retinol dehydrogenase RDH8 to all-trans-Retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-Retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-Retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for Retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 μm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to Retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and Retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.
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Formation of all-trans Retinol after visual pigment bleaching in mouse photoreceptors.
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Chunhe Chen, Lorie R. Blakeley, Yiannis KoutalosAbstract:Vision is initiated by the absorption of light by the visual pigment present in the outer segments of the rod and cone photoreceptor cells in the retina. In both cell types, the first step in the detection of light is the photoisomerization of the retinyl chromophore of the visual pigment from 11-cis to all-trans.1,2 This isomerization of the chromophore bleaches the pigment, necessitating its regeneration with fresh 11-cis retinal. The production of 11-cis retinal includes the recycling of the all-trans chromophore of the bleached pigment through a series of reactions called the visual cycle.3–5 These reactions begin in the outer segment with the release of all-trans retinal from the photoactivated pigment and its reduction to all-trans Retinol by Retinol dehydrogenase in a reaction using NADPH.6 The all-trans Retinol is then transferred from outer segments to the adjacent retinal pigment epithelial cells,7 where it is esterified to form retinyl ester.8,9 The ester is converted to 11-cis Retinol,10–13 which is then oxidized to 11-cis retinal.14 Studies of cone-dominant ground squirrel and chicken retinas have provided evidence of the presence of an additional visual cycle used by cones.15–17 Continuous vision depends on the regeneration of the visual pigment and defects in the processing of the chromophore through the reactions of the visual cycle are responsible for a wide range of visual defects.3,18 Extensive studies using whole eyes have argued for the presence of two slow steps in the operation of the mouse visual cycle: the formation of all-trans Retinol and the isomerization of all-trans retinyl ester.19–21 Although the formation of all-trans Retinol has been characterized in detail in amphibian photoreceptors,22–24 these results cannot provide a quantitative insight into the operation of the mouse visual cycle because of critical species differences, such as body temperature. Therefore, we undertook the measurement of the kinetics of all-trans Retinol formation in mouse photoreceptors by HPLC of retinoid extracts and fluorescence imaging. In our results, all-trans Retinol formation was not the slowest step in the mouse visual cycle, which supports the notion that the isomerization of retinyl esters is the slowest step.20,21 Throughout the text, unqualified retinal and Retinol refer to the all-trans isomers.
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Formation Of All-Trans Retinol In Mouse Rod Photoreceptors
Biophysical Journal, 2009Co-Authors: Lorie R. Blakeley, Chunhe Chen, Yiannis KoutalosAbstract:Light detection destroys the visual pigment of vertebrate rod photoreceptors, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. Rhodopsin is regenerated through a series of reactions that begin in the rod outer segment with the release of the all-trans retinal and its reduction to all-trans Retinol. All-trans Retinol is then transported to the neighboring retinal pigment epithelial cells where it is used to remake 11-cis retinal. The reduction of all-trans retinal to all-trans Retinol is catalyzed by Retinol dehydrogenase and requires metabolic input in the form of NADPH. We have used the fluorescence of all-trans Retinol to monitor its concentration in isolated mouse rod photoreceptors. After the bleaching of rhodopsin, all-trans Retinol formation proceeds with a rate of ∼0.06 min−1, which is faster than the rate of rhodopsin regeneration in whole animals; this would allow recycled chromophore to contribute to the 11-cis retinal used for regeneration. Inner segment metabolic pathways appear to make a significant contribution to the pool of NADPH needed for the reduction of all-trans retinal, as formation of all-trans Retinol is suppressed in rod outer segments separated from the cell body. Finally, generation of all-trans Retinol is suppressed in the absence of glucose, indicating a critical dependence of all-trans Retinol formation on the level of metabolic activity.
