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William S Blaner - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the Acyltransferase activity of Lecithin Retinol Acyltransferase like proteins
Journal of Biological Chemistry, 2012Co-Authors: Marcin Golczak, William S Blaner, Philip D Kiser, Avery E Sears, David T Lodowski, Krzysztof PalczewskiAbstract:Lecithin:Retinol Acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to Lecithin:Retinol Acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent Acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.
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diethylnitrosamine induced hepatocarcinogenesis is suppressed in Lecithin Retinol Acyltransferase deficient mice primarily through retinoid actions immediately after carcinogen administration
Carcinogenesis, 2012Co-Authors: Yohei Shirakami, Max E Gottesman, William S BlanerAbstract:Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and Lecithin:Retinol Acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.
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metabolic basis of visual cycle inhibition by retinoid and nonretinoid compounds in the vertebrate retina
Journal of Biological Chemistry, 2008Co-Authors: Marcin Golczak, Grzegorz Bereta, Johannes Von Lintig, Philip D Kiser, Akiko Maeda, Silke Hunzelmann, Tadao Maeda, William S BlanerAbstract: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.
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retinoid absorption and storage is impaired in mice lacking Lecithin Retinol Acyltransferase lrat
Journal of Biological Chemistry, 2005Co-Authors: Sheila M Obyrne, Wolfgang Baehr, Krzysztof Palczewski, Nuttaporn Wongsiriroj, Silke Vogel, Jenny Libien, Ira J Goldberg, William S BlanerAbstract:Abstract Lecithin:Retinol Acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of Retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for Retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic Retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary Retinol primarily as free Retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these chylomicron retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:Retinol Acyltransferase.
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Cellular Retinol-binding protein type III is needed for retinoid incorporation into milk.
Journal of Biological Chemistry, 2005Co-Authors: Roseann Piantedosi, William S Blaner, Norbert Ghyselinck, Silke VogelAbstract:The physiologic role(s) of cellular Retinol-binding protein (CRBP)-III, an intracellular Retinol-binding protein that is expressed solely in heart, muscle, adipose, and mammary tissue, remains to be elucidated. To address this, we have generated and characterized CRBP-III-deficient (CRBP-III(-/-)) mice. Mice that lack CRBP-III were viable and healthy but displayed a marked impairment in retinoid incorporation into milk. Milk obtained from CRBP-III(-/-) dams contains significantly less retinyl ester, especially retinyl palmitate, than milk obtained from wild type dams. We demonstrated that Retinol bound to CRBP-III is an excellent substrate for Lecithin-Retinol Acyltransferase, the enzyme responsible for catalyzing retinyl ester formation from Retinol. Our data indicated that the diminished milk retinyl ester levels arise from impaired utilization of Retinol by Lecithin-Retinol Acyltransferase in CRBP-III(-/-) mice. Interestingly, CRBP-I and CRBP-III each appeared to compensate for the absence of the other, specifically in mammary tissue, adipose tissue, muscle, and heart. For CRBP-III(-/-) mice, CRBP-I protein levels were markedly elevated in adipose tissue and mammary gland. In addition, in CRBP-I(-/-) mice, CRBP-III protein levels were elevated in tissues that normally express CRBP-III but were not elevated in other tissues that do not normally express CRBP-III. Our data suggested that CRBP-I and CRBP-III share some physiologic actions within tissues and that each can compensate for the absence of the other to help maintain normal retinoid homeostasis. However, under conditions of high demand for retinoid, such as those experienced during lactation, this compensation was incomplete.
L Aravind - One of the best experts on this subject based on the ideXlab platform.
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evolutionary history structural features and biochemical diversity of the nlpc p60 superfamily of enzymes
Genome Biology, 2003Co-Authors: Vivek Anantharaman, L AravindAbstract:Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-γ-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages. Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include Lecithin Retinol Acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.
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Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes
Genome Biology, 2003Co-Authors: Vivek Anantharaman, L AravindAbstract:Background Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D -γ-glutamyl-meso-diaminopimelate or N -acetylmuramate-L-alanine linkages. Results Detailed analysis of the N1pC/P60 peptidases showed that these proteins define a large superfamily encompassing several diverse groups of proteins. In addition to the well characterized P60-like proteins, this superfamily includes the AcmB/LytN and YaeF/YiiX families of bacterial proteins, the amidase domain of bacterial and kinetoplastid glutathionylspermidine synthases (GSPSs), and several proteins from eukaryotes, phages, poxviruses, positive-strand RNA viruses, and certain archaea. The eukaryotic members include Lecithin Retinol Acyltransferase (LRAT), nematode developmental regulator Egl-26, and candidate tumor suppressor H-rev107. These eukaryotic proteins, along with the bacterial YaeF/poxviral G6R family, show a circular permutation of the catalytic domain. We identified three conserved residues, namely a cysteine, a histidine and a polar residue, that are involved in the catalytic activities of this superfamily. Evolutionary analysis of this superfamily shows that it comprises four major families, with diverse domain architectures in each of them. Conclusions Several related, but distinct, catalytic activities, such as murein degradation, acyl transfer and amide hydrolysis, have emerged in the N1pC/P60 superfamily. The three conserved catalytic residues of this superfamily are shown to be equivalent to the catalytic triad of the papain-like thiol peptidases. The predicted structural features indicate that the N1pC/P60 enzymes contain a fold similar to the papain-like peptidases, transglutaminases and arylamine acetyltransferases.
Natsuo Ueda - One of the best experts on this subject based on the ideXlab platform.
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the tumor suppressor gene h rev107 functions as a novel ca2 independent cytosolic phospholipase a1 2 of the thiol hydrolase type
Journal of Lipid Research, 2009Co-Authors: Toru Uyama, Jun Morishita, Xinghua Jin, Yasuo Okamoto, Kazuhito Tsuboi, Natsuo UedaAbstract:H-Rev107 is a protein that was previously cloned as a negative regulator of proto-oncogene Ras and classified as a class II tumor suppressor. Its structural similarity to Lecithin Retinol Acyltransferase and Ca2+-independent phosphatidylethanolamine (PE) N-Acyltransferase led us to analyze H-Rev107 as an enzyme involved in phospholipid metabolism. Here, we show that recombinant H-Rev107s from rat, human, and mouse possess phospholipase (PL) A1 or A2 activity toward phosphatidylcholine (PC). Further examination with purified recombinant protein revealed that H-Rev107 functions as a cytosolic Ca2+-independent PLA1/2 for PC and PE with higher PLA1 activity than PLA2 activity. Dithiothreitol and iodoacetic acid exhibited stimulatory and inhibitory effects, respectively. Histidine-21 and cysteine-111 of rat H-Rev107 were presumed to form a catalytic dyad based on database analysis, and their single mutants were totally inactive. These results suggested that H-Rev107 is a hydrolase of the thiol type. The N-terminal proline-rich and C-terminal hydrophobic domains of H-Rev107 were earlier reported to be responsible for the regulation of cell proliferation. Analysis of deletion mutants indicated that these domains are also catalytically essential, suggesting relevance of the catalytic activity to the anti-proliferative activity.
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discovery and characterization of a ca2 independent phosphatidylethanolamine n Acyltransferase generating the anandamide precursor and its congeners
Journal of Biological Chemistry, 2006Co-Authors: Xinghua Jin, Jun Morishita, Yasuo Okamoto, Kazuhito Tsuboi, Takeharu Tonai, Natsuo UedaAbstract:Abstract N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-Acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to Lecithin-Retinol Acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylcholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-Acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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Lecithin Retinol Acyltransferase is critical for cellular uptake of vitamin a from serum Retinol binding protein
Journal of Biological Chemistry, 2012Co-Authors: Jaume Amengual, Krzysztof Palczewski, Marcin Golczak, Johannes Von LintigAbstract:Vitamin A (all-trans-Retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to Retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular Lecithin:Retinol Acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat−/− mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating Retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.
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structural basis for the Acyltransferase activity of Lecithin Retinol Acyltransferase like proteins
Journal of Biological Chemistry, 2012Co-Authors: Marcin Golczak, William S Blaner, Philip D Kiser, Avery E Sears, David T Lodowski, Krzysztof PalczewskiAbstract:Lecithin:Retinol Acyltransferase-like proteins, also referred to as HRAS-like tumor suppressors, comprise a vertebrate subfamily of papain-like or NlpC/P60 thiol proteases that function as phospholipid-metabolizing enzymes. HRAS-like tumor suppressor 3, a representative member of this group, plays a key role in regulating triglyceride accumulation and energy expenditure in adipocytes and therefore constitutes a novel pharmacological target for treatment of metabolic disorders causing obesity. Here, we delineate a catalytic mechanism common to Lecithin:Retinol Acyltransferase-like proteins and provide evidence for their alternative robust lipid-dependent Acyltransferase enzymatic activity. We also determined high resolution crystal structures of HRAS-like tumor suppressor 2 and 3 to gain insight into their active site architecture. Based on this structural analysis, two conformational states of the catalytic Cys-113 were identified that differ in reactivity and thus could define the catalytic properties of these two proteins. Finally, these structures provide a model for the topology of these enzymes and allow identification of the protein-lipid bilayer interface. This study contributes to the enzymatic and structural understanding of HRAS-like tumor suppressor enzymes.
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retinoid absorption and storage is impaired in mice lacking Lecithin Retinol Acyltransferase lrat
Journal of Biological Chemistry, 2005Co-Authors: Sheila M Obyrne, Wolfgang Baehr, Krzysztof Palczewski, Nuttaporn Wongsiriroj, Silke Vogel, Jenny Libien, Ira J Goldberg, William S BlanerAbstract:Abstract Lecithin:Retinol Acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of Retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for Retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic Retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary Retinol primarily as free Retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these chylomicron retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:Retinol Acyltransferase.
Jian Xing - One of the best experts on this subject based on the ideXlab platform.
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α phenyl n tert butylnitrone pbn prevents light induced degeneration of the retina by inhibiting rpe65 protein isomerohydrolase activity
Journal of Biological Chemistry, 2011Co-Authors: Nawajes A Mandal, Gennadiy Moiseyev, Jian Xing, Olga Nikolaeva, Michael H Elliott, Anne Kasusjacobi, Hui Chen, Lixin Zheng, Robert A Floyd, Robert E AndersonAbstract:α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5–12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC50 = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with Ki = 53 μm. PBN had no effect on the activity of other visual cycle enzymes, Lecithin Retinol Acyltransferase and Retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.
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inhibition of the visual cycle by a2e through direct interaction with rpe65 and implications in stargardt disease
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Gennadiy Moiseyev, Yusuke Takahashi, Ying Chen, Olga Nikolaeva, Krysten M Farjo, Jian XingAbstract:Stargardt disease (STGD) is the major form of inherited juvenile macular degeneration. Pyridinium bis-retinoid A2E is a major component of lipofuscin which accumulates in retinal pigment epithelium (RPE) cells in STGD and contributes to the disease pathogenesis. However, the precise role of A2E in vision loss is unclear. Here we report that A2E efficiently inhibits RPE65 isomerohydrolase, a key enzyme in the visual cycle. Subretinal injection of A2E significantly inhibited retinoid isomerohydrolase activity in mice. Likewise, A2E also inhibited isomerohydrolase activity in cells coexpressing RPE65, Lecithin Retinol Acyltransferase (LRAT), and cellular retinaldehyde-binding protein. In vitro isomerohydrolase activity assays confirmed that A2E inhibited enzymatic activity of recombinant RPE65 in a concentration-dependent manner, but did not inhibit LRAT activity. The inhibition type for isomerohydrolase was found to be reversible and competitive with Ki = 13.6 μM. To determine the direct interaction of A2E with RPE65 protein, fluorescence binding assays were performed. As shown by fluorimetric titration, binding of purified RPE65 with A2E enhanced the bis-retinoid fluorescence. Consistently, the fluorescence of RPE65 decreased upon incubation with A2E. Both of these experiments suggest a direct, specific binding of A2E to RPE65. The binding constant for A2E and purified RPE65 was calculated to be 250 nM. These results demonstrate that A2E inhibits the regeneration of 11-cis retinal, the chromophore of visual pigments, which represents a unique mechanism by which A2E may impair vision in STGD.
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the 11 cis Retinol dehydrogenase activity of rdh10 and its interaction with visual cycle proteins
Investigative Ophthalmology & Visual Science, 2009Co-Authors: Krysten M Farjo, Yusuke Takahashi, Gennadiy Moiseyev, Rosalie K Crouch, Jian XingAbstract:PURPOSE. The final step in the retinoid visual cycle is catalyzed by 11-cis-Retinol dehydrogenases (11-cis-RDHs) that oxidize 11cis-Retinol (11cROL) to 11-cis-retinaldehyde (11cRAL). Genetic studies in mice indicate that the full repertoire of 11-cis-RDH enzymes remains to be identified. This study was conducted to characterize the 11-cis-RDH activity of RDH10 in vitro and specifically to determine whether RDH10 can functionally and physically interact with visual cycle proteins. METHODS. Human RDH10 was expressed in COS1 cells to measure its 11-cis-RDH activity in the presence or absence of purified recombinant cellular retinaldehyde-binding protein (CRALBP). The RPE visual cycle was reconstituted in HEK-293A cells by co-expressing RDH10, CRALBP, RPE-specific 65-kDa protein (RPE65) and Lecithin Retinol Acyltransferase (LRAT). The cells were subsequently treated with all-trans-Retinol (atROL), and retinoid profiles were quantified by HPLC. Immunocytochemical and co-immunoprecipitation analyses were performed to determine whether RDH10 physically interacts with other visual cycle proteins. RESULTS. RDH10 oxidized 11cROL to generate 11cRAL in vitro in the presence of CRALBP. RDH10 can use both NAD and NADP as cofactors for 11-cis-RDH activity, although NAD cofactor confers more robust activity. In a cell culture model co-expressing RDH10 with RPE65, LRAT and CRALBP, the visual chromophore 11cRAL was generated from atROL. Immunohistochemistry showed that RDH10 co-localizes with RPE65 and CRALBP in vivo in primary bovine RPE cells. Immunoprecipitation analysis demonstrated that RDH10 physically interacts with CRALBP and RPE65. CONCLUSIONS. RDH10 may function in the RPE retinoid visual cycle as an 11-cis-RDH, and thereby partially compensate for the loss of RDH5 function in patients with fundus albipunctatus. (Invest Ophthalmol Vis Sci. 2009;50:5089‐5097) DOI:10.1167/iovs.09-3797
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RPE65 is the isomerohydrolase in the retinoid visual cycle
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Gennadiy Moiseyev, Yusuke Takahashi, Ying Chen, Jian XingAbstract: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.