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1.
Proc Natl Acad Sci U S A ; 119(11): e2115202119, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35271391

RESUMO

SignificanceIn humans, genetic mutations in the retinal pigment epithelium (RPE) 65 are associated with blinding diseases, for which there is no effective therapy alleviating progressive retinal degeneration in affected patients. Our findings uncovered that the increased free opsin caused by enhancing the ambient light intensity increased retinal activation, and when compounded with the RPE visual cycle dysfunction caused by the heterozygous D477G mutation and aggregation, led to the onset of retinal degeneration.


Assuntos
Proteínas do Olho , Genes Dominantes , Distrofias Retinianas , cis-trans-Isomerases , Animais , Proteínas do Olho/genética , Camundongos , Camundongos Knockout , Mutação , Retina/enzimologia , Retina/patologia , Distrofias Retinianas/genética , Visão Ocular , cis-trans-Isomerases/genética
2.
Adv Exp Med Biol ; 1415: 415-419, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440066

RESUMO

The retina pigmented epithelium 65 kDa protein (RPE65) is an essential enzyme in the visual cycle that regenerates the 11-cis-retinal chromophore obligatory for vision. Mutations in RPE65 are associated with blinding diseases. D477G (C.1430G > A) is the only known RPE65 variant to cause autosomal dominant retinitis pigmentosa (adRP). Previously, we reported that the heterozygous D477G knock-in (WT/KI) mice exposed to dim light intensity demonstrated delayed chromophore regeneration rates and slowed recovery of photoreceptor sensitivity following photobleaching. However, visual function and retinal architecture were indistinguishable from the wild-type (WT) mice. In this study, when maintained under the physiological day-light intensity (2 K lux), the WT/KI heterozygous mice displayed retina degeneration and reduced electroretinography (ERG) amplitude, recapitulating that observed in human patients. Our findings indicated the importance of the light environment in the mechanism of RPE65 D477G pathogenicity.


Assuntos
Degeneração Retiniana , cis-trans-Isomerases , Humanos , Camundongos , Animais , Modelos Animais de Doenças , cis-trans-Isomerases/genética , Retina/metabolismo , Mutação , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Epitélio
3.
Am J Pathol ; 190(7): 1505-1512, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32275905

RESUMO

Diabetic retinopathy (DR) is a common complication of diabetes and a leading cause of blindness among the working-age population. Diabetic patients often experience functional deficits in dark adaptation, contrast sensitivity, and color perception before any microvascular pathologies on the fundus become detectable. Previous studies showed that the regeneration of 11-cis-retinal and visual pigment is impaired in a type 1 diabetes animal model, which negatively affects visual function at the early stage of DR. Here, Akita mice, type 1 diabetic model, were treated with the visual pigment chromophore, 9-cis-retinal. This treatment rescued a- and b-wave amplitudes of scotopic electroretinography responses, compared with vehicle-treated Akita mice. In addition, the administration of 9-cis-retinal alleviated oxidative stress significantly as shown by reduced 3-nitrotyrosine levels in the retina of Akita mice. Furthermore, the 9-cis-retinal treatment decreased retinal apoptosis as shown by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and DNA fragment enzyme-linked immunosorbent assay. Overall, these findings showed that 9-cis-retinal administration restored visual pigment formation and decreased oxidative stress and retinal degeneration, which resulted in improved visual function in diabetic mice, suggesting that chromophore deficiency plays a causative role in visual defects in early DR.


Assuntos
Retinopatia Diabética/fisiopatologia , Diterpenos/farmacologia , Retina/efeitos dos fármacos , Retinaldeído/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Estresse Oxidativo/efeitos dos fármacos , Retina/fisiopatologia
4.
Biochim Biophys Acta Mol Basis Dis ; 1864(7): 2420-2429, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29684583

RESUMO

The retinoid visual cycle is an ocular retinoid metabolism specifically dedicated to support vertebrate vision. The visual cycle serves not only to generate light-sensitive visual chromophore 11-cis-retinal, but also to clear toxic byproducts of normal visual cycle (i.e. all-trans-retinal and its condensation products) from the retina, ensuring both the visual function and the retinal health. Unfortunately, various conditions including genetic predisposition, environment and aging may attribute to a functional decline of the all-trans-retinal clearance. To combat all-trans-retinal mediated retinal degeneration, we sought to slow down the retinoid influx from the RPE by inhibiting the visual cycle with a small molecule. The present study describes identification of CU239, a novel non-retinoid inhibitor of RPE65, a key enzyme in the visual cycle. Our data demonstrated that CU239 selectively inhibited isomerase activity of RPE65, with IC50 of 6 µM. Further, our results indicated that CU239 inhibited RPE65 via competition with its substrate all-trans-retinyl ester. Mice with systemic injection of CU239 exhibited delayed chromophore regeneration after light bleach, and conferred a partial protection of the retina against injury from high intensity light. Taken together, CU239 is a potent visual cycle modulator and may have a therapeutic potential for retinal degeneration.


Assuntos
Inibidores Enzimáticos/farmacologia , Degeneração Retiniana , Visão Ocular , cis-trans-Isomerases , Animais , Diterpenos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/enzimologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Ésteres de Retinil , Visão Ocular/efeitos dos fármacos , Visão Ocular/genética , Vitamina A/análogos & derivados , Vitamina A/metabolismo , cis-trans-Isomerases/antagonistas & inibidores , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
5.
Am J Pathol ; 187(3): 517-527, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28041994

RESUMO

RPE65 is an indispensable component of the retinoid visual cycle in vertebrates, through which the visual chromophore 11-cis-retinal (11-cis-RAL) is generated to maintain normal vision. Various blinding conditions in humans, such as Leber congenital amaurosis and retinitis pigmentosa (RP), are attributed to either homozygous or compound heterozygous mutations in RPE65. Herein, we investigated D477G missense mutation, an unprecedented dominant-acting mutation of RPE65 identified in patients with autosomal dominant RP. We generated a D477G knock-in (KI) mouse and characterized its phenotypes. Although RPE65 protein levels were decreased in heterozygous KI mice, their scotopic, maximal, and photopic electroretinography responses were comparable to those of wild-type (WT) mice in stationary condition. As shown by high-performance liquid chromatography analysis, levels of 11-cis-RAL in fully dark-adapted heterozygous KI mice were similar to that in WT mice. However, kinetics of 11-cis-RAL regeneration after light exposure were significantly slower in heterozygous KI mice compared with WT and RPE65 heterozygous knockout mice. Furthermore, heterozygous KI mice exhibited lower A-wave recovery compared with WT mice after photobleaching, suggesting a delayed dark adaptation. Taken together, these observations suggest that D477G acts as a dominant-negative mutant of RPE65 that delays chromophore regeneration. The KI mice provide a useful model for further understanding of the pathogenesis of RP associated with this RPE65 mutant and for the development of therapeutic strategies.


Assuntos
Adaptação à Escuridão/genética , Técnicas de Introdução de Genes , Genes Dominantes , Mutação/genética , Vias Visuais/metabolismo , cis-trans-Isomerases/genética , Animais , Cromatografia Líquida de Alta Pressão , Eletrorretinografia , Heterozigoto , Isomerases/metabolismo , Camundongos Mutantes , Modelos Animais , Opsinas/metabolismo , Fotodegradação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Retina/metabolismo , Retina/patologia , Retinoides/metabolismo , cis-trans-Isomerases/metabolismo
6.
Am J Pathol ; 187(10): 2222-2231, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28734946

RESUMO

Diabetic retinopathy is a common complication of diabetes mellitus. Diabetic patients experience functional deficits in dark adaptation, contrast sensitivity, and color perception before microvascular pathologies become apparent. Herein, we evaluated early changes in neural retinal function and in retinoid metabolism in the eye in diabetes. Streptozotocin-induced diabetic rats showed decreased a- and b-wave amplitudes of scotopic and photopic electroretinography responses 4 months after diabetes induction compared to nondiabetic controls. Although Western blot analysis revealed no difference in opsin expression, rhodopsin content was decreased in diabetic retinas, as shown by a difference in absorbance. Consistently, levels of 11-cis-retinal, the chromophore for visual pigments, were significantly lower in diabetic retinas compared to those in controls, suggesting a retinoid deficiency. Among visual cycle proteins, interphotoreceptor retinoid-binding protein and stimulated by retinoic acid 6 protein showed significantly lower levels in diabetic rats than those in nondiabetic controls. Similarly, serum levels of retinol-binding protein 4 and retinoids were significantly lower in diabetic rats. Overall, these results suggest that retinoid metabolism in the eye is impaired in type 1 diabetes, which leads to deficient generation of visual pigments and neural retinal dysfunction in early diabetes.


Assuntos
Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Rodopsina/metabolismo , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/sangue , Retinopatia Diabética/complicações , Modelos Animais de Doenças , Masculino , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos Wistar , Retina/patologia , Retina/fisiopatologia , Retinaldeído/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Vias Visuais/metabolismo , Vias Visuais/patologia
7.
Kidney Int ; 91(3): 642-657, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27914705

RESUMO

Pigment epithelium-derived factor (PEDF) expression is downregulated in the kidneys of diabetic rats, and delivery of PEDF suppressed renal fibrotic factors in these animals. PEDF has multiple functions including anti-angiogenic, anti-inflammatory and antifibrotic activities. Since the mechanism underlying its antifibrotic effect remains unclear, we studied this in several murine models of renal disease. Renal PEDF levels were significantly reduced in genetic models of type 1 and type 2 diabetes (Akita and db/db, respectively), negatively correlating with Wnt signaling activity in the kidneys. In unilateral ureteral obstruction, an acute renal injury model, there were significant decreases of renal PEDF levels. The kidneys of PEDF knockout mice with ureteral obstruction displayed exacerbated expression of fibrotic and inflammatory factors, oxidative stress, tubulointerstitial fibrosis, and tubule epithelial cell apoptosis, compared to the kidneys of wild-type mice with obstruction. PEDF knockout enhanced Wnt signaling activation induced by obstruction, while PEDF inhibited the Wnt pathway-mediated fibrosis in primary renal proximal tubule epithelial cells. Additionally, oxidative stress was aggravated in renal proximal tubule epithelial cells isolated from knockout mice and suppressed by PEDF treatment of renal proximal tubule epithelial cells. PEDF also reduced oxidation-induced apoptosis in renal proximal tubule epithelial cells. Thus, the renoprotective effects of PEDF are mediated, at least partially, by inhibition of the Wnt pathway. Hence, restoration of renal PEDF levels may have therapeutic potential for renal fibrosis.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Nefropatias/prevenção & controle , Túbulos Renais Proximais/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Obstrução Ureteral/metabolismo , Via de Sinalização Wnt , Animais , Apoptose , Proteína Axina/genética , Proteína Axina/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Células Epiteliais/patologia , Proteínas do Olho/genética , Fibrose , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Crescimento Neural/deficiência , Fatores de Crescimento Neural/genética , Estresse Oxidativo , Fenótipo , Serpinas/deficiência , Serpinas/genética , Fatores de Tempo , Transfecção , Obstrução Ureteral/complicações , Obstrução Ureteral/genética , Obstrução Ureteral/patologia
8.
J Biol Chem ; 289(39): 26743-26751, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25112876

RESUMO

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 (Met(1)-Arg(33)) 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.


Assuntos
Mutação de Sentido Incorreto , cis-trans-Isomerases , Substituição de Aminoácidos , Animais , Bovinos , Linhagem Celular , Galinhas , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Terapia Genética , Humanos , Mutagênese Sítio-Dirigida , Degeneração Retiniana/enzimologia , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Especificidade da Espécie , Relação Estrutura-Atividade , cis-trans-Isomerases/química , cis-trans-Isomerases/imunologia , cis-trans-Isomerases/metabolismo
9.
J Biol Chem ; 287(29): 24092-102, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22645143

RESUMO

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.


Assuntos
Fator Inibidor de Leucemia/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Eletrorretinografia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
10.
Proc Natl Acad Sci U S A ; 107(41): 17551-6, 2010 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-20876139

RESUMO

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 K(i) = 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.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas do Olho/antagonistas & inibidores , Degeneração Macular/metabolismo , Compostos de Piridínio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Visão Ocular/fisiologia , Aciltransferases/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Fluorescência , Camundongos , Ligação Proteica , Compostos de Piridínio/farmacologia , Retinoides/farmacologia , cis-trans-Isomerases
11.
Cell Rep ; 42(2): 112091, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36763501

RESUMO

Retinosomes are intracellular lipid bodies found in the retinal pigment epithelium (RPE). They contain retinyl esters (REs) and are thought to be involved in visual chromophore regeneration during dark adaptation and in case of chromophore depletion. However, key enzymes in chromophore regeneration, retinoid isomerase (RPE65), and lecithin:retinol acyltransferase (LRAT) are located in the endoplasmic reticulum (ER). The mechanism and the enzyme responsible for mobilizing REs from retinosomes remained unknown. Our study demonstrates that patatin-like phospholipase domain containing 2 (PNPLA2) mobilizes all-trans-REs from retinosomes. The absence of PNPLA2 in mouse eyes leads to a significant accumulation of lipid droplets in RPE cells, declined electroretinography (ERG) response, and delayed dark adaptation compared with those of WT control mouse. Our work suggests a function of PNPLA2 as an RE hydrolase in the RPE, mobilizing REs from lipid bodies and functioning as an essential component of the visual cycle.


Assuntos
Retinaldeído , Ésteres de Retinil , Animais , Camundongos , Eletrorretinografia , Epitélio Pigmentado da Retina , Vitamina A
12.
Invest Ophthalmol Vis Sci ; 64(11): 33, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37642632

RESUMO

Purpose: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. Methods: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. Results: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. Conclusions: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models.


Assuntos
Córnea , Respiração , Animais , Camundongos , Camundongos Endogâmicos C57BL , Biópsia , Trifosfato de Adenosina , Mitocôndrias
13.
Biochemistry ; 51(20): 4217-25, 2012 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-22512451

RESUMO

The efficient recycling of the chromophore of visual pigments, 11-cis-retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis-retinol (11cROL) and a minor amount of 13-cis-retinol (13cROL), from all-trans-retinyl ester (atRE). We recently identified and characterized novel homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining the isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103, and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.


Assuntos
Domínio Catalítico , Peixe-Zebra/metabolismo , cis-trans-Isomerases/química , Sequência de Aminoácidos , Animais , Galinhas , Leucina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Epitélio Pigmentado Ocular/metabolismo , Especificidade por Substrato , Tirosina/química , Vitamina A/metabolismo , cis-trans-Isomerases/genética
14.
Dev Biol ; 357(2): 347-55, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21782811

RESUMO

Retinoic acid (atRA) signaling is essential for regulating embryonic development, and atRA levels must be tightly controlled in order to prevent congenital abnormalities and fetal death which can result from both excessive and insufficient atRA signaling. Cellular enzymes synthesize atRA from Vitamin A, which is obtained from dietary sources. Embryos express multiple enzymes that are biochemically capable of catalyzing the initial step of Vitamin A oxidation, but the precise contribution of these enzymes to embryonic atRA synthesis remains unknown. Using Rdh10(trex)-mutant embryos, dietary supplementation of retinaldehyde, and retinol dehydrogenase (RDH) activity assays, we demonstrate that RDH10 is the primary RDH responsible for the first step of embryonic Vitamin A oxidation. Moreover, we show that this initial step of atRA synthesis occurs predominantly in a membrane-bound cellular compartment, which prevents inhibition by the cytosolic cellular retinol-binding protein (RBP1). These studies reveal that widely expressed cytosolic enzymes with RDH activity play a very limited role in embryonic atRA synthesis under normal dietary conditions. This provides a breakthrough in understanding the precise cellular mechanisms that regulate Vitamin A metabolism and the synthesis of the essential embryonic regulatory molecule atRA.


Assuntos
Oxirredutases do Álcool/metabolismo , Embrião de Mamíferos/metabolismo , Tretinoína/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Oxirredução , Fosfolipídeos/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismo , Especificidade por Substrato
15.
J Biol Chem ; 286(37): 32491-501, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21785167

RESUMO

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 IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 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.


Assuntos
Proteínas de Transporte/metabolismo , Óxidos N-Cíclicos/farmacologia , Proteínas do Olho/metabolismo , Luz/efeitos adversos , Fármacos Neuroprotetores/farmacologia , Degeneração Retiniana , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Rodopsina/metabolismo , cis-trans-Isomerases/metabolismo , Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Animais , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/enzimologia , Degeneração Retiniana/prevenção & controle
16.
Biochem J ; 436(3): 591-7, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21446919

RESUMO

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 atRE (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 atRE, 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 atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Proteínas do Olho/metabolismo , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , cis-trans-Isomerases/metabolismo , Animais , Hidrolases de Éster Carboxílico/química , Galinhas , Diterpenos , Proteínas do Olho/química , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Ésteres de Retinil , Vitamina A/análogos & derivados , Vitamina A/metabolismo , cis-trans-Isomerases/química
17.
PLoS One ; 17(10): e0269437, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36227868

RESUMO

The visual cycle refers to a series of biochemical reactions of retinoids in ocular tissues and supports the vision in vertebrates. The visual cycle regenerates visual pigments chromophore, 11-cis-retinal, and eliminates its toxic byproducts from the retina, supporting visual function and retinal neuron survival. Unfortunately, during the visual cycle, when 11-cis-retinal is being regenerated in the retina, toxic byproducts, such as all-trans-retinal and bis-retinoid is N-retinylidene-N-retinylethanolamine (A2E), are produced, which are proposed to contribute to the pathogenesis of the dry form of age-related macular degeneration (AMD). The primary biochemical defect in Stargardt disease (STGD1) is the accelerated synthesis of cytotoxic lipofuscin bisretinoids, such as A2E, in the retinal pigment epithelium (RPE) due to mutations in the ABCA4 gene. To prevent all-trans-retinal-and bisretinoid-mediated retinal degeneration, slowing down the retinoid flow by modulating the visual cycle with a small molecule has been proposed as a therapeutic strategy. The present study describes RPE65-61, a novel, non-retinoid compound, as an inhibitor of RPE65 (a key enzyme in the visual cycle), intended to modulate the excessive activity of the visual cycle to protect the retina from harm degenerative diseases. Our data demonstrated that (±)-RPE65-61 selectively inhibited retinoid isomerase activity of RPE65, with an IC50 of 80 nM. Furthermore, (±)-RPE65-61 inhibited RPE65 via an uncompetitive mechanism. Systemic administration of (±)-RPE65-61 in mice resulted in slower chromophore regeneration after light bleach, confirming in vivo target engagement and visual cycle modulation. Concomitant protection of the mouse retina from high-intensity light damage was also observed. Furthermore, RPE65-61 down-regulated the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway, decreased the inflammatory factor, and attenuated retinal apoptosis caused by light-induced retinal damage (LIRD), which led to the preservation of the retinal function. Taken together, (±)-RPE65-61 is a potent visual cycle modulator that may provide a neuroprotective therapeutic benefit for patients with STGD and AMD.


Assuntos
Degeneração Macular , Degeneração Retiniana , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Interferons/metabolismo , Lipofuscina/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologia , Degeneração Retiniana/prevenção & controle , Pigmentos da Retina/metabolismo , Retinaldeído/metabolismo , Retinaldeído/farmacologia , Retinoides/metabolismo , Retinoides/farmacologia , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismo
18.
Diabetes ; 70(3): 788-799, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33334874

RESUMO

Patients with diabetes often experience visual defects before any retinal pathologies are detected. The molecular mechanism for the visual defects in early diabetes has not been elucidated. Our previous study reported that in early diabetic retinopathy (DR), rhodopsin levels were reduced due to impaired 11-cis-retinal regeneration. Interphotoreceptor retinol-binding protein (IRBP) is a visual cycle protein and important for 11-cis-retinal generation. IRBP levels are decreased in the vitreous and retina of DR patients and animal models. To determine the role of IRBP downregulation in the visual defects in early DR, we induced diabetes in transgenic mice overexpressing IRBP in the retina. IRBP overexpression prevented diabetes-induced decline of retinal function. Furthermore, IRBP overexpression also prevented decreases of rhodopsin levels and 11-cis-retinal generation in diabetic mice. Diabetic IRBP transgenic mice also showed ameliorated retinal oxidative stress, inflammation, apoptosis, and retinal degeneration compared with diabetic wild-type mice. These findings suggest that diabetes-induced IRBP downregulation impairs the regeneration of 11-cis-retinal and rhodopsin, leading to retinal dysfunction in early DR. Furthermore, increased 11-cis-retinal-free opsin constitutively activates the phototransduction pathway, leading to increased oxidative stress and retinal neurodegeneration. Therefore, restored IRBP expression in the diabetic retina may confer a protective effect against retinal degeneration in DR.


Assuntos
Retinopatia Diabética/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Rodopsina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Retinopatia Diabética/genética , Proteínas do Olho/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Proteínas de Ligação ao Retinol/genética , Rodopsina/genética , Tomografia de Coerência Óptica
19.
Biochem Biophys Res Commun ; 391(4): 1757-61, 2010 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-20043869

RESUMO

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Ácido Glutâmico/metabolismo , Amaurose Congênita de Leber/enzimologia , cis-trans-Isomerases/metabolismo , Cegueira/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas do Olho/química , Proteínas do Olho/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Humanos , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Eletricidade Estática , cis-trans-Isomerases/química , cis-trans-Isomerases/genética
20.
Biochem J ; 419(1): 113-22, 1 p following 122, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19102727

RESUMO

RDH10 (retinol dehydrogenase 10) was originally identified from the retinal pigment epithelium and retinal Müller cells. It has retinoid oxidoreductase activity and is thought to play a role in the retinoid visual cycle. A recent study showed that RDH10 is essential for generating retinoic acid at early embryonic stages. The present study demonstrated that wild-type RDH10 catalysed both oxidation of all-trans-retinol and reduction of all-trans-retinal in a cofactor-dependent manner In vitro. In cultured cells, however, oxidation is the favoured reaction catalysed by RDH10. Substitution of any of the predicted key residues in the catalytic centre conserved in the RDH family abolished the enzymatic activity of RDH10 without affecting its protein level. Unlike other RDH members, however, replacement of Ser(197), a key residue for stabilizing the substrate, by glycine and alanine did not abolish the enzymatic activity of RDH10, whereas RDH10 mutants S197C, S197T and S197V completely lost their enzymatic activity. These results suggest that the size of the residue at position 197 is critical for the activity of RDH10. Mutations of the three glycine residues (Gly(43), Gly(47) and Gly(49)) in the predicted cofactor-binding motif (Gly-Xaa(3)-Gly-Xaa-Gly) of RDH10 abolished its enzymatic activity, suggesting that the cofactor-binding motif is essential for its activity. Deletion of the two hydrophobic domains dissociated RDH10 from the membrane and abolished its activity. These studies identified the key residues for the activity of RDH10 and will contribute to the further elucidation of mechanism of this important enzyme.


Assuntos
Oxirredutases do Álcool/metabolismo , Membrana Celular/metabolismo , Estrutura Terciária de Proteína/fisiologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Estrutura Terciária de Proteína/genética , Ratos , Homologia de Sequência de Aminoácidos
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