The Experts below are selected from a list of 50364 Experts worldwide ranked by ideXlab platform
Jian Xing - One of the best experts on this subject based on the ideXlab platform.
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Identification of key residues determining isomerohydrolase activity of human RPE65.
The Journal of biological chemistry, 2014Co-Authors: Yusuke Takahashi, Gennadiy Moiseyev, Jian XingAbstract:RPE65 is the retinoid isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, a key reaction in the retinoid visual cycle. We have previously reported that cone-dominant chicken RPE65 (cRPE65) shares 90% sequence identity with human RPE65 (hRPE65) but exhibits substantially higher isomerohydrolase activity than that of bovine RPE65 or hRPE65. In this study, we sought to identify key residues responsible for the higher enzymatic activity of cRPE65. Based on the amino acid sequence comparison of mammalian and other lower vertebrates' RPE65, including cone-dominant chicken, 8 residues of hRPE65 were separately replaced by their counterparts of cRPE65 using site-directed mutagenesis. The enzymatic activities of cRPE65, hRPE65, and its mutants were measured by in vitro isomerohydrolase activity assay, and the retinoid products were analyzed by HPLC. Among the mutants analyzed, two single point mutants, N170K and K297G, and a double mutant, N170K/K297G, of hRPE65 exhibited significantly higher catalytic activity than WT hRPE65. Further, when an amino-terminal fragment (Met1–Arg33) of the N170K/K297G double mutant of hRPE65 was replaced with the corresponding cRPE65 fragment, the isomerohydrolase activity was further increased to a level similar to that of cRPE65. This finding contributes to the understanding of the structural basis for isomerohydrolase activity. This highly efficient human isomerohydrolase mutant can be used to improve the efficacy of RPE65 gene therapy for retinal degeneration caused by RPE65 mutations.
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RPE65 Is Present in Human Green/Red Cones and Promotes Photopigment Regeneration in an In Vitro Cone Cell Model
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011Co-Authors: Peter H. Tang, Jian Xing, Mona Buhusi, Rosalie K. CrouchAbstract:RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.
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RPE65 is present in human green red cones and promotes photopigment regeneration in an in vitro cone cell model
The Journal of Neuroscience, 2011Co-Authors: Peter H. Tang, Jian Xing, Mona Buhusi, Rosalie K. CrouchAbstract:RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.
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Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity.
The Biochemical journal, 2011Co-Authors: Olga Nikolaeva, Gennadiy Moiseyev, Karla K. Rodgers, Jian XingAbstract:The visual cycle is a multi-step pathway to recycle 11- cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts a t RE (all- trans -retinyl ester) to 11- cis -retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of a t RE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing a t RE. These results demonstrated that RPE659s functional activity depends on its conformational changes caused by its association with the membrane.
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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.
T. Michael Redmond - One of the best experts on this subject based on the ideXlab platform.
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RPE65 Palmitoylation: A Tale of Lipid Posttranslational Modification
Advances in experimental medicine and biology, 2019Co-Authors: Sheetal Uppal, Eugenia Poliakov, Susan Gentleman, T. Michael RedmondAbstract:RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65’s membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.
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Inhibition of RPE65 Retinol Isomerase Activity by Inhibitors of Lipid Metabolism
The Journal of biological chemistry, 2015Co-Authors: Abdulkerim Eroglu, Susan Gentleman, Eugenia Poliakov, T. Michael RedmondAbstract:RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.
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The RPE65 rd12 allele exerts a semidominant negative effect on vision in mice.
Investigative ophthalmology & visual science, 2014Co-Authors: Charles B. Wright, T. Michael Redmond, Micah A Chrenek, Todd Duncan, Machelle T. Pardue, Jeffrey H. Boatright, Wei Feng, Shannon Getz, Yue Feng, John M. NickersonAbstract:Purpose The rd12 mouse was reported as a recessively inherited RPE65 mutation. We asked if the rd12 mutation resides in RPE65 and how the mutation manifests itself. Methods A complementation test was performed by mating RPE65(KO) (KO/KO) and rd12 mice together to determine if the rd12 mutation is in the RPE65 gene. Visual function of wild-type (+/+), KO/+, rd12/+, KO/KO, rd12/rd12, and KO/rd12 mice was measured by optokinetic tracking (OKT) and ERG. Morphology was assessed by retinal cross section. qRT-PCR quantified RPE65 mRNA levels. Immunoblotting measured the size and level of RPE65 protein. RPE65 mRNA localization was visualized with RNA fluorescence in situ hybridization (FISH). Fractions of RPE65 mRNA-bound proteins were separated by linear sucrose gradient fractionation. Results The KO and rd12 alleles did not complement. The rd12 allele induced a negative semidominant effect on visual function; OKT responses became undetectable 120 days earlier in rd12/rd12 mice compared with KO/KO mice. rd12/+ mice lost approximately 21% visual acuity by P210. rd12/rd12 mice had fewer cone photoreceptor nuclei than KO/KO mice at P60. rd12/rd12 mice expressed 71% +/+ levels of RPE65 mRNA, but protein was undetectable. Mutant mRNA was appropriately spliced, exported to the cytoplasm, trafficked, and contained no other coding mutation aside from the known nonsense mutation. Mutant mRNA was enriched on ribosome-free messenger ribonucleoproteins (mRNPs), whereas wild-type mRNA was enriched on actively translating polyribosomes. Conclusions The rd12 lesion is in RPE65. The rd12 mutant phenotype inherits in a semidominant manner. The effects of the mutant mRNA on visual function may result from inefficient binding to ribosomes for translation.
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Complementation Test of RPE65 Knockout and Tvrm148
Investigative ophthalmology & visual science, 2013Co-Authors: Charles B. Wright, T. Michael Redmond, Micah A Chrenek, Stephanie L. Foster, Todd Duncan, Machelle T. Pardue, Jeffrey H. Boatright, John M. NickersonAbstract:Purpose A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and RPE65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if RPE65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels. Methods Normal phase HPLC was used to measure retinoid levels. RPE65(+/+), tvrm148/+ (T(+/-)), tvrm148/tvrm148 (T(-/-)), RPE65(KO/KO) (RPE65(-/-)), and RPE65(T/-) mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure RPE65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein. Results The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T(-/-) or RPE65(-/-) mice. Visual acuity in RPE65(+/+) and T(+/-) mouse was -0.382 c/d, but 0.037 c/d in T(-/-) mice at postnatal day 210 (P210). ERG response in T(-/-) mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T(-/-) mice was -70% of RPE65(+/+) by P210. RPE65 mRNA levels in T(-/-) mice were unchanged, yet 14.5% of RPE65(+/+) protein levels was detected. Protein size was unchanged. Conclusions A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in RPE65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of RPE65.
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Aromatic lipophilic spin traps effectively inhibit RPE65 isomerohydrolase activity
Biochemistry, 2011Co-Authors: Eugenia Poliakov, Susan Gentleman, Toral Parikh, Michael Ayele, Stephanie Kuo, Preethi Chander, T. Michael RedmondAbstract:We previously showed that RPE65 does not specifically produce 11-cis-retinol only but also 13-cis-retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-α-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO), and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro.
Rosalie K. Crouch - One of the best experts on this subject based on the ideXlab platform.
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RPE65 and the Accumulation of Retinyl Esters in Mouse Retinal Pigment Epithelium
Photochemistry and Photobiology, 2017Co-Authors: Colleen Sheridan, Nicholas P. Boyer, Rosalie K. Crouch, Yiannis KoutalosAbstract:The RPE65 protein of the retinal pigment epithelium (RPE) enables the conversion of retinyl esters to the visual pigment chromophore 11-cis retinal. Fresh 11-cis retinal is generated from retinyl esters following photoisomerization of the visual pigment chromophore to all-trans during light detection. Large amounts of esters accumulate in RPE65-/- mice, indicating their continuous formation when 11-cis retinal generation is blocked. We hypothesized that absence of light, by limiting the conversion of esters to 11-cis retinal, would also result in the build-up of retinyl esters in the RPE of wild-type mice. We used HPLC to quantify ester levels in organic extracts of the RPE from wild-type and RPE65-/- mice. Retinyl ester levels in Sv/129 wild-type mice that were dark adapted for various intervals over a 4-week period were similar to those in mice raised in cyclic light. In C57BL/6 mice however, which contain less RPE65 protein, dark adaptation was accompanied by an increase in ester levels compared to cyclic light controls. Retinyl ester levels were much higher in RPE65-/- mice compared to wild type and kept increasing with age. The results suggest that the RPE65 role in retinyl ester homeostasis extends beyond enabling the formation of 11-cis retinal.
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RPE65 is present in human green red cones and promotes photopigment regeneration in an in vitro cone cell model
The Journal of Neuroscience, 2011Co-Authors: Peter H. Tang, Jian Xing, Mona Buhusi, Rosalie K. CrouchAbstract:RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.
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RPE65 Is Present in Human Green/Red Cones and Promotes Photopigment Regeneration in an In Vitro Cone Cell Model
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011Co-Authors: Peter H. Tang, Jian Xing, Mona Buhusi, Rosalie K. CrouchAbstract:RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.
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Deletion of GRK1 Causes Retina Degeneration through a Transducin-Independent Mechanism
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2010Co-Authors: Jie Fan, Keisuke Sakurai, Ching-kang Chen, Baerbel Rohrer, King-wai Yau, Vladimir J. Kefalov, Rosalie K. CrouchAbstract:RPE65 −/− mice are unable to produce 11- cis -retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9- cis -retinal as chromophore. Notably, a 10–20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the RPE65 −/− Grk1 −/− mouse. The retinae of RPE65 −/− Grk1 −/− mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to RPE65 −/− mice. These data show that opsin phosphorylation in the RPE65 −/− mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating RPE65 −/− Grk1 −/− mice resulted in lower overall opsin activity and in higher rod sensitivity compared with RPE65 −/− mice. In RPE65 −/− Grk1 −/− Gnat1 −/− mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1 −/− mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1 −/− Gnat1 −/− mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.
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Identification of a Novel Palmitylation Site Essential for Membrane Association and Isomerohydrolase Activity of RPE65
The Journal of biological chemistry, 2008Co-Authors: Yusuke Takahashi, Rosalie K. Crouch, Gennadiy Moiseyev, Ying Chen, Zsolt Ablonczy, Jian XingAbstract:RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.
Samuel G. Jacobson - One of the best experts on this subject based on the ideXlab platform.
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Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins
Advances in experimental medicine and biology, 2015Co-Authors: Minghao Jin, Heather H. Jin, Samuel G. Jacobson, Dean BokAbstract:More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.
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rescue of enzymatic function for disease associated RPE65 proteins containing various missense mutations in non active sites
Journal of Biological Chemistry, 2014Co-Authors: Tadahide Izumi, Heather H. Jin, Samuel G. Jacobson, Dean Bok, Ahmedabdul A Siddiqui, Minghao JinAbstract:Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway. These mutant RPE65s were misfolded and formed aggregates or high molecular complexes via disulfide bonds. Interaction of PSMD13 with mutant RPE65s promoted degradation of misfolded but not properly folded mutant RPE65s. Many mutations, including L22P, T101I, and L408P, were mapped on non-active sites. Although their activities were very low, these mutant RPE65s were catalytically active and could be significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. Sodium 4-phenylbutyrate and glycerol displayed a significant synergistic effect on the low temperature rescue of the mutant RPE65s by promoting proper folding, reducing aggregation, and increasing membrane association. Our results suggest that a low temperature eye mask and sodium 4-phenylbutyrate, a United States Food and Drug Administration-approved oral medicine, may provide a promising “protein repair therapy” that can enhance the efficacy of gene therapy by reducing the cytotoxic effect of misfolded mutant RPE65s.
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human retinal gene therapy for leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Artur V Cideciyan, Samuel G. Jacobson, Sharon B Schwartz, Alejandro J. Roman, Alexander Sumaroka, William A Beltran, Malgorzata Swider, Simone Iwabe, Melani B Olivares, Andras M KomaromyAbstract:Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5–11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term.
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the usher 1b protein myo7a is required for normal localization and function of the visual retinoid cycle enzyme RPE65
Human Molecular Genetics, 2011Co-Authors: Vanda S Lopes, Tomas S Aleman, Samuel G. Jacobson, Roxana A. Radu, Daniel Gibbs, Richard T Libby, Darcy L Welch, Concepcion Lillo, Karen P SteelAbstract:Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B. In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place. We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle. We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells. This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light. RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels. Following a 50–60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity. Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction. Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.
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Retinal disease in RPE65-deficient mice: comparison to human leber congenital amaurosis due to RPE65 mutations.
Investigative ophthalmology & visual science, 2010Co-Authors: Rafael C. Caruso, Tomas S Aleman, Artur V Cideciyan, William W Hauswirth, Sanford L Boye, Alejandro J. Roman, Alexander Sumaroka, Cristina L. Mullins, Samuel G. JacobsonAbstract:PURPOSE To quantify the retinal disease in RPE65-deficient mice across a wide age span and compare the results to those in humans with Leber congenital amaurosis (LCA) caused by RPE65 mutations. METHODS Full-field electroretinograms (ERGs) were recorded from wild-type (C57BL/6; RPE65(+/+)) and RPE65(-/-) mice at ages ranging from ∼1 month to 2 years. A physiologically based model of rod phototransduction activation was used to determine photoreceptor (P3) cell components of ERG photoresponses. A bipolar (P2) cell component was also derived. Photoreceptor and inner retinal thickness measurements were made by using optical coherence tomography in human RPE65-LCA. RESULTS Age-related declines in ERG photoreceptor and bipolar amplitudes were present in the RPE65(-/-) mouse. The loss of photoresponse amplitude with age in the mutant mice paralleled reported losses of photoreceptor nuclear layer thickness over the same age range. Unexpectedly, the early activation phase of photoresponses in RPE65(-/-) mice accelerated with age as amplitude decreased; this was not a feature of RPE65(+/+) mice. Inner retinal dysfunction increased with age in the mutant mice. Human RPE65-LCA patients had retinal degeneration and loss of photoreceptors in the first decade of life. Unlike the mouse model, there were no examples of a normal photoreceptor complement. Abnormal thickening of the inner retina occurred with increasing loss of photoreceptors. CONCLUSIONS The differences in time course of murine and human RPE65-deficiency diseases suggests that preclinical efficacy testing of therapeutic modalities would be most informative when the murine disease becomes comparable to early human disease, toward the end of the first year of life in RPE65(-/-) mice.
Artur V Cideciyan - One of the best experts on this subject based on the ideXlab platform.
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human retinal gene therapy for leber congenital amaurosis shows advancing retinal degeneration despite enduring visual improvement
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Artur V Cideciyan, Samuel G. Jacobson, Sharon B Schwartz, Alejandro J. Roman, Alexander Sumaroka, William A Beltran, Malgorzata Swider, Simone Iwabe, Melani B Olivares, Andras M KomaromyAbstract:Leber congenital amaurosis (LCA) associated with retinal pigment epithelium-specific protein 65 kDa (RPE65) mutations is a severe hereditary blindness resulting from both dysfunction and degeneration of photoreceptors. Clinical trials with gene augmentation therapy have shown partial reversal of the dysfunction, but the effects on the degeneration are not known. We evaluated the consequences of gene therapy on retinal degeneration in patients with RPE65-LCA and its canine model. In untreated RPE65-LCA patients, there was dysfunction and degeneration of photoreceptors, even at the earliest ages. Examined serially over years, the outer photoreceptor nuclear layer showed progressive thinning. Treated RPE65-LCA showed substantial visual improvement in the short term and no detectable decline from this new level over the long term. However, retinal degeneration continued to progress unabated. In RPE65-mutant dogs, the first one-quarter of their lifespan showed only dysfunction, and there was normal outer photoreceptor nuclear layer thickness retina-wide. Dogs treated during the earlier dysfunction-only stage showed improved visual function and dramatic protection of treated photoreceptors from degeneration when measured 5–11 y later. Dogs treated later during the combined dysfunction and degeneration stage also showed visual function improvement, but photoreceptor loss continued unabated, the same as in human RPE65-LCA. The results suggest that, in RPE65 disease treatment, protection from visual function deterioration cannot be assumed to imply protection from degeneration. The effects of gene augmentation therapy are complex and suggest a need for a combinatorial strategy in RPE65-LCA to not only improve function in the short term but also slow retinal degeneration in the long term.
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Retinal disease in RPE65-deficient mice: comparison to human leber congenital amaurosis due to RPE65 mutations.
Investigative ophthalmology & visual science, 2010Co-Authors: Rafael C. Caruso, Tomas S Aleman, Artur V Cideciyan, William W Hauswirth, Sanford L Boye, Alejandro J. Roman, Alexander Sumaroka, Cristina L. Mullins, Samuel G. JacobsonAbstract:PURPOSE To quantify the retinal disease in RPE65-deficient mice across a wide age span and compare the results to those in humans with Leber congenital amaurosis (LCA) caused by RPE65 mutations. METHODS Full-field electroretinograms (ERGs) were recorded from wild-type (C57BL/6; RPE65(+/+)) and RPE65(-/-) mice at ages ranging from ∼1 month to 2 years. A physiologically based model of rod phototransduction activation was used to determine photoreceptor (P3) cell components of ERG photoresponses. A bipolar (P2) cell component was also derived. Photoreceptor and inner retinal thickness measurements were made by using optical coherence tomography in human RPE65-LCA. RESULTS Age-related declines in ERG photoreceptor and bipolar amplitudes were present in the RPE65(-/-) mouse. The loss of photoresponse amplitude with age in the mutant mice paralleled reported losses of photoreceptor nuclear layer thickness over the same age range. Unexpectedly, the early activation phase of photoresponses in RPE65(-/-) mice accelerated with age as amplitude decreased; this was not a feature of RPE65(+/+) mice. Inner retinal dysfunction increased with age in the mutant mice. Human RPE65-LCA patients had retinal degeneration and loss of photoreceptors in the first decade of life. Unlike the mouse model, there were no examples of a normal photoreceptor complement. Abnormal thickening of the inner retina occurred with increasing loss of photoreceptors. CONCLUSIONS The differences in time course of murine and human RPE65-deficiency diseases suggests that preclinical efficacy testing of therapeutic modalities would be most informative when the murine disease becomes comparable to early human disease, toward the end of the first year of life in RPE65(-/-) mice.
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Defining the residual vision in leber congenital amaurosis caused by RPE65 mutations.
Investigative ophthalmology & visual science, 2008Co-Authors: Samuel G. Jacobson, Tomas S Aleman, Artur V Cideciyan, Sharon B Schwartz, Alejandro J. Roman, Alexander Sumaroka, Elise Héon, Elizabeth A. M. Windsor, Edwin M. StoneAbstract:Leber congenital amaurosis (LCA) is a molecularly heterogeneous group of autosomal recessive diseases with early onset of severe visual loss.1 One molecular form of LCA is due to mutations in the gene encoding RPE65 (retinal pigment epithelium-specific 65 kDa protein). Scientific interest in RPE65 is high because this molecule is the long-sought isomerohydrolase of the visual cycle.2–4 Medical interest has also been piqued by proof-of-concept experiments showing restored vision in two species of RPE65-deficient animals by subretinal gene replacement therapy5–10 and now in human clinical trials.11–14 Despite this remarkable progress, human RPE65-LCA disease remains incompletely understood. We know that severely impaired vision is associated with inheritance of two RPE65 alleles which show little or no isomerohydrolase activity in vitro.15 A visual toll is taken on both central and peripheral vision and on both rod and cone photoreceptor-mediated function (reviewed in Ref. 16). Unlike RPE65-deficient animal models at early ages and stages, children with early visual dysfunction also have a serious retinal degeneration.9,17,18 The retinal degeneration component tends to be masked by dysfunction resulting from the visual cycle abnormality, but eventually there is clinically apparent pigmentary retinopathy and chorioretinal atrophy.16 Adding to the complexity is the fact that the visual cycle blockade in humans and animals is obviously not complete, because there is residual vision. Animal models with RPE65 deficiency have residual photoreceptor function.5,6,19–21 In mutant mice, the residual function has been ascribed to either low levels of production of 11-cis-retinal or RPE65-independent pathways.21,22 The goal of the present study was to determine quantitatively key features of the residual vision in a population of patients with RPE65-LCA. We inquired whether the residual function was only cone- or also rod-mediated by using electroretinography (ERG) and full-field sensitivity testing (FST). The pattern of visual loss across the retina was explored with kinetic and dark-adapted static perimetry. The results have implications, not only for understanding the human disease but also for enhancing strategies for therapy of RPE65-LCA.
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treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno associated virus gene vector short term results of a phase i trial
Human Gene Therapy, 2008Co-Authors: William W Hauswirth, Tomas S Aleman, Artur V Cideciyan, Shalesh Kaushal, Sharon B Schwartz, Lili Wang, Thomas J Conlon, Sanford L Boye, Terence R Flotte, Barry J ByrneAbstract:Abstract Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21–24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 × 1010 vector genomes in 150 μl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic tox...
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Photoreceptor Layer Topography in Children with Leber Congenital Amaurosis Caused by RPE65 Mutations
Investigative ophthalmology & visual science, 2008Co-Authors: Samuel G. Jacobson, Tomas S Aleman, Artur V Cideciyan, Sharon B Schwartz, Alexander Sumaroka, Elise Héon, Elizabeth A. M. Windsor, Edwin M. StoneAbstract:Human clinical trials of subretinal gene replacement therapy in Leber congenital amaurosis (LCA) due to mutations in the gene encoding RPE65 (retinal pigment epithelium-specific 65-kDa protein) are ongoing in different centers.1–4 Current trials are open-label uniocular single subretinal injections of adeno-associated viral vector altered to carry the human RPE65 gene. Studies are being conducted first in small cohorts of adult subjects. In two recent reports of preliminary results in patients ranging in age from 17 to 26 years of age, the authors indicate an intention to advance to cohorts of children with RPE65-LCA5–7 What do we know about the retinal disease in children with RPE65-LCA? Normal photoreceptor histology in models of RPE65-LCA in young mice and dogs (for example, Refs. 8,9) could lead to an assumption that patients with RPE65-LCA may also have normal or near normal photoreceptor counts early in life. Human RPE65-LCA, however, is not only a biochemical dysfunction of the visual cycle but also a serious retinal degeneration that is present, even in the first decade of life.10–12 This important difference between young humans and young disease models of RPE65-LCA has therapeutic implications. Children with better-preserved photoreceptors and RPE cells, for example, should in theory have greater potential for therapeutic efficacy. Experimental support for this logical concept was provided in proof-of-concept studies that compared RPE65 −/− mice at young and older ages.10 Mutant mice at later stages of disease (i.e., with considerable photoreceptor loss) showed less restoration of retinal function and retinoid biochemistry after subretinal gene replacement therapy than did younger mice.10 Cross-sectional retinal imaging with optical coherence tomography (OCT) provides the opportunity to determine non-invasively in humans the photoreceptor cell layer thickness in a wide expanse of central retina.10 We studied photoreceptor layer thickness in young patients with RPE65-LCA to inquire whether there were retinal locations with a higher likelihood of retained photoreceptors and thereby more suitable as loci for subretinal injections of vector-gene product. There was variation between subjects in the degree of photoreceptor layer integrity, but summary maps of photoreceptor loss revealed consistent patterns.
