RESUMO
Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4-/- mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4+/- , and KI;Fatp4-/- mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4-/- mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.
Assuntos
Proteínas de Transporte de Ácido Graxo/deficiência , Amaurose Congênita de Leber/fisiopatologia , Retina/patologia , Visão Ocular/fisiologia , cis-trans-Isomerases/genética , Animais , Opsinas dos Cones/metabolismo , Modelos Animais de Doenças , Diterpenos/isolamento & purificação , Proteínas de Transporte de Ácido Graxo/genética , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Camundongos , Camundongos Knockout , Mutação , Retina/metabolismo , Retinaldeído/biossíntese , Retinaldeído/isolamento & purificação , cis-trans-Isomerases/metabolismoRESUMO
Opsin expression is extremely suppressed by carotenoid deprivation in Drosophila. Carotenoid replacement in deprived flies promotes the recovery of visual pigment with an increase in opsin, as well as the chromophore 11-cis-3-hydroxyretinal. Here, we show that opsin mRNA and opsin peptide in an intermediate step of posttranslational processing were present in carotenoid-deprived flies. By supplementing chromophore to photoreceptor cells, intermediate opsin was made mature. During this process, opsin peptide underwent multiple modifications involving glycosylation. Based on these results, we present a novel mechanism of protein regulatory expression; that is, chromophore posttranslationally controls the expression of apoprotein by promoting its maturation.
Assuntos
Drosophila melanogaster/fisiologia , Retinaldeído/análogos & derivados , Rodopsina/biossíntese , Opsinas de Bastonetes/biossíntese , Animais , Expressão Gênica , Glicosilação , Immunoblotting , Células Fotorreceptoras/fisiologia , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Retinaldeído/isolamento & purificação , Retinaldeído/metabolismo , Rodopsina/isolamento & purificação , Opsinas de Bastonetes/isolamento & purificação , Transcrição GênicaRESUMO
Visual pigments are a class of receptor proteins that absorb light and trigger sensory signals. Retinal-containing proteins are used in nature as photoreceptors mainly in animals vision. Mammalian rhodopsin is the best studied example of a light sensor which couples photon absorption to a cascade of biochemical reactions amplifying the input signal. A surprising discovery was to find rhodopsin also in Archaebacteria and in unicellular eukaryotes. On the basis of absorption microspectroscopic measurements and of inhibition experiments on pigment biosynthetic pathways, we have recently suggested that a rhodopsin could be the functional receptor of the visual process in Euglena gracilis, a flagellate which can use light directly to promote photosynthetic reactions, or as an incident flux of information to adjust its swimming orientation. We here report purification and identification of all-trans-retinal by column chromatography, HPLC and GC-MS in E. gracilis; these findings indicate with absolute certainty that rhodopsin is the photoreceptor molecule of this microorganism.
Assuntos
Euglena gracilis/química , Retinaldeído/isolamento & purificação , Rodopsina/isolamento & purificação , Animais , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Retinaldeído/química , Rodopsina/química , Espectrofotometria UltravioletaRESUMO
The bioluminescent squid, Watasenia scintillans has three visual pigments. The major pigment, based on retinal (lambda max 484 nm), is distributed over the whole retina. Another pigment based on 3-dehydroretinal (lambda max approximately 500 nm) and the third pigment (lambda max approximately 470 nm) are localized in the specific area of the ventral retina just receiving the downwelling light. Visual pigment was extracted and purified from the dissected retina. The chromophores were then extracted and analyzed with HPLC, NMR, infrared and mass spectroscopy, being compared with the synthetic 4-hydroxyretinal. A new retinal derivative, 11-cis-4-hydroxyretinal, is identified as the chromophore of the third visual pigment of the squid.
Assuntos
Decapodiformes/análise , Retinaldeído/análogos & derivados , Retinaldeído/isolamento & purificação , Retinoides/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão , Diterpenos , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Retina/análise , Retinaldeído/análise , Espectrofotometria UltravioletaRESUMO
Enzymatic conversion of all-trans-beta-carotene to retinal by a partially purified enzyme from rabbit, rat, and human neonatal intestinal mucosa has been demonstrated. The enzymatic product was characterized based on the following evidence. First, the product gave rise to its O-ethyl oxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal (O-ethyl) oxime. High-performance liquid chromatography of this derivative yielded a sharp peak with a retention time of 7.99 min, corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal (O-ethyl) oxime or retinal or retinol. Second, the mass spectrum of the O-ethyl oxime of the enzymatic product was identical to that of authentic retinal (O-ethyl) oxime (m/z 327, 45%; m+ and m/z 282, 100%, methoxy). Third, the 14C radioactivity persisted to constant specific activity even after repeated crystallization of the retinal (O-ethyl) oxime isolated from the enzyme reaction with purified beta-[14C]carotene. Fourth, the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC, corresponding to authentic retinyl palmitate. Thus, the enzymatic product of beta-carotene cleavage by the partially purified intestinal enzyme has been unequivocally confirmed to be retinal. Similarly, enzymatic conversion of all-trans-beta-carotene to retinal by an intestinal mucosal enzyme from autopsy samples of human neonates has also been demonstrated. Based on the observed activities among intestinal samples from 12 premature infants, the BCC enzyme activity ranged from 3.3 to 1210 pmol/mg mucosal protein/hr. However, the observed activities in the human autopsy samples may be markedly underestimated, presumably because of marked loss of enzyme activity from the time of death to the time of assay. Therefore, the true activity of the enzyme can be assessed only after the extent of the loss of its activity on storage of the human samples can be accurately measured. Nonetheless, the demonstration of BCC enzyme activity in human neonates shows that beta-carotene may be an important source of vitamin A nutrition during gestation.
Assuntos
Carotenoides/metabolismo , Mucosa Intestinal/enzimologia , Oxigenases/metabolismo , Retinaldeído/metabolismo , Animais , Biotransformação , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão/métodos , Cristalização , Humanos , Indicadores e Reagentes , Recém-Nascido , Recém-Nascido Prematuro , Espectrometria de Massas , Oxigenases/isolamento & purificação , Coelhos , Ratos , Retinaldeído/isolamento & purificação , Estereoisomerismo , beta Caroteno , beta-Caroteno 15,15'-Mono-OxigenaseRESUMO
7-cis Retinal, one of the geometrical isomers of retinal, was prepared by the direct irradiation of all-trans retinal dissolved in ethanol and successive separation by high performance liquid chromatography. The production for its purification and identification are described.
Assuntos
Luz , Retinaldeído/efeitos da radiação , Vitamina A/análogos & derivados , Animais , Apoproteínas , Bovinos , Cromatografia Líquida de Alta Pressão , Conformação Molecular/efeitos da radiação , Retinaldeído/isolamento & purificação , Rodopsina , Análise Espectral , EstereoisomerismoRESUMO
The chromophore of visual pigments was efficiently extracted in its original isomeric conformation by dichloromethane/hexane in the presence of excess formaldehyde. This new method of extraction (the formaldehyde method) in combination with HPLC is useful for the determination of the chromophore composition of visual pigments. The chromophore isolated by this method is biologically active and can be used for regeneration experiments.
Assuntos
Pigmentos da Retina/isolamento & purificação , Animais , Astacoidea , Bovinos , Cromatografia Líquida de Alta Pressão , Formaldeído , Métodos , Oximas , Retinaldeído/isolamento & purificação , Segmento Externo da Célula Bastonete/análise , TemperaturaRESUMO
Bacteriorhodopsin (bR) is an attractive intelligent material. Understanding the mechanism of its light-driven proton pumping outward the cell implicates it in many technical applications, particularly, in what is called optical computers, and the biotechnology is waiting for this promised biological molecule. An ionizing radiation source handling could be computerized in radiation fields. The computer containing such biological material will not be out of reach of the fields of ionizing radiation. So it is interesting to report on the working of such biological computer if it is subjected to ionizing radiation. The functional unit in this molecule is retinal chromophore. In the present work, it is interested to assess the functionality of bR through determining the electronic transition dipole moment of its chromophore. Significant changes in the values of the absorption transition dipole moment were noticed at different doses of beta-particles in the range of 0.1-0.3 kGy. Ionizing radiation-induced changes in bR were followed by intrinsic fluorescence spectroscopy. An analysis of the fluorescence data bears on the tertiary structure of bR. The emission spectrum is, however, red shifted with an increase in intensity with the different doses; in the meanwhile, gradual decrease in the visible absorbance has occurred till almost complete loss is attained. This bleaching due to ionizing radiation may offer an alternative way of data processing in such optical devices based on bR. Nevertheless, bR has proofed to be used as a biological indicator of ionizing radiation. However, the potential of bR for use as a biosensor to detect ionizing radiation should be considered.
Assuntos
Bacteriorodopsinas/efeitos da radiação , Partículas beta , Halobacterium salinarum/química , Retinaldeído/efeitos da radiação , Bacteriorodopsinas/análise , Bacteriorodopsinas/isolamento & purificação , Biotecnologia , Relação Dose-Resposta à Radiação , Matemática , Fotoquímica , Bombas de Próton , Membrana Purpúrea/química , Monitoramento de Radiação , Retinaldeído/análise , Retinaldeído/isolamento & purificação , Bases de Schiff , Triptofano/química , Tirosina/químicaRESUMO
The isolation of the retinal isomers from all-trans-retinal was performed by flash countercurrent chromatography. In each separation, isomerization reaction solution of 200mg all-trans-retinal could be loaded on a 1200 mL of high-speed countercurrent chromatographic column with 5mm bore, eluted by a mobile phase flow rate of 25 mL/min, resulting in 63 mg of 11-cis-retinal, 24 mg of 13-cis-retinal and 26 mg of 9-cis-retinal with purities more than 95%. n-Hexane-acetonitrile (3:1) was used as the solvent system which possesses the advantages of simplicity, re-use of the solvent and multiple injections. This method could be used to prepare 13-cis-retinal, 11-cis-retinal and 9-cis-retinal for the photoisomerization investigation, such as the effect of 11-cis-retinal in the visual system.
Assuntos
Distribuição Contracorrente/métodos , Retinaldeído/isolamento & purificação , Vitamina A/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Isomerismo , Retinaldeído/química , Vitamina A/químicaRESUMO
Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimer's disease, type 2 diabetes, obesity, and cancer.
Assuntos
Retinoides/análise , Métodos Analíticos de Preparação de Amostras , Animais , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Proteínas do Olho/análise , Proteínas do Olho/isolamento & purificação , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Lineares , Masculino , Camundongos , Oxirredução , Padrões de Referência , Reprodutibilidade dos Testes , Retinaldeído/química , Retinaldeído/isolamento & purificação , Retinoides/sangue , Retinoides/química , Retinoides/isolamento & purificação , Manejo de Espécimes , Espectrofotometria Ultravioleta , Frações Subcelulares/química , Espectrometria de Massas em Tandem , Vitamina A/química , Vitamina A/isolamento & purificaçãoRESUMO
The retinoid (visual) cycle is a complex enzymatic pathway essential for regeneration of the visual chromophore, 11-cis-retinal, a component of rhodopsin that undergoes activation by light in vertebrate eyes. Pathogenic mutations within genes encoding proteins involved in the retinoid cycle lead to abnormalities in retinoid homeostasis and numerous congenital blinding diseases of humans. Thus, elucidation of disease-specific changes in enzymatic activities and retinoid content of the retina can provide important insights into the mechanisms of disease initiation and progression. Here, we use the protein RPE65 as an example to describe generally applicable methods for determining the stability and enzymatic activity of proteins and their mutants involved in retinoid metabolism. Additionally, we introduce a range of analytical techniques involving high-performance liquid chromatography and mass spectrometry to detect and quantify retinoids and their derivatives in eye extracts. Biochemical protocols combined with advanced mass spectrometry should facilitate fundamental biological studies of vision.
Assuntos
Técnicas de Química Analítica/métodos , Biologia Molecular/métodos , Retinoides/química , Retinoides/metabolismo , Aciltransferases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Ésteres , Olho/metabolismo , Proteínas do Olho/metabolismo , Isomerismo , Lipofuscina/metabolismo , Espectrometria de Massas , Camundongos , Células NIH 3T3 , Compostos de Piridínio/metabolismo , Retinaldeído/análogos & derivados , Retinaldeído/química , Retinaldeído/isolamento & purificação , Retinaldeído/metabolismo , Retinoides/análise , Retinoides/isolamento & purificação , Vitamina A/química , Vitamina A/isolamento & purificação , Vitamina A/metabolismo , cis-trans-IsomerasesAssuntos
Carotenoides/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Citosol/enzimologia , Cinética , Oxigenases/metabolismo , Técnica de Diluição de Radioisótopos , Ratos , Retinaldeído/isolamento & purificação , Especificidade por Substrato , Trítio , beta Caroteno , beta-Caroteno 15,15'-Mono-OxigenaseAssuntos
Proteínas de Transporte/isolamento & purificação , Retina/análise , Retinaldeído/isolamento & purificação , Proteínas de Ligação ao Retinol/isolamento & purificação , Tretinoína/isolamento & purificação , Vitamina A/análogos & derivados , Animais , Bovinos , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , Peso Molecular , Ligação Proteica , Receptores do Ácido Retinoico , Retinaldeído/metabolismo , Tretinoína/metabolismo , Vitamina A/metabolismoAssuntos
Abelhas/análise , Pigmentos da Retina/isolamento & purificação , Animais , Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Olho/análise , Peso Molecular , Retinaldeído/isolamento & purificação , Proteínas de Ligação ao Retinol/isolamento & purificação , Espectrofotometria , EstereoisomerismoAssuntos
Cyprinidae , Carpa Dourada , Epitélio Pigmentado Ocular/análise , Vitamina A/análise , Animais , Cromatografia Líquida de Alta Pressão , Adaptação à Escuridão , Ésteres/isolamento & purificação , Oximas/isolamento & purificação , Retinaldeído/isolamento & purificação , Vitamina A/análogos & derivadosRESUMO
The light-dark adaptation reactions of a set of bacteriorhodopsin (bR) mutants that affect function and color of the chromophore were examined by using visible absorption spectroscopy. The absorbance spectra of the mutants Arg-82 in equilibrium Ala (Gln), Asp-85 in equilibrium Ala (Asn, Glu), Tyr-185 in equilibrium Phe, and Asp-212 in equilibrium Ala (Asn, Glu) were measured at different pH values during and after illumination. None of these mutants exhibited a normal dark-light adaptation, which in wild-type bR causes a red shift of the visible absorption maximum from 558 nm (dark-adapted bR) to 568 nm (light-adapted bR). Instead a reversible light reaction occurs in the Asp-85 and Asp-212 mutants from a blue form with lambda max near 600 nm to a pink form with lambda max near 480 nm. This light-induced shift explains the appearance of a reversed light adaptation previously observed for the Asp-212 mutants. In the case of the Tyr-185 and Arg-82 mutants, light causes a purple-to-blue transformation similar to the effect of lowering the pH. However, the blue forms observed in these mutants are not identical to those formed by acid titration or deionization of wild-type bR. It is suggested that in all of these mutants, the chromophore has lost the ability to undergo the normal 13-cis, 15-syn to all-trans, 15-anti light-driven isomerization, which occurs in native bR. Instead these mutants may have as stable forms all-trans,syn and 13-cis,anti chromophores, which are not allowed in native bR, except transiently.