RPE apical proteins that regulate the visual cycle

调节视觉周期的 RPE 顶端蛋白

• 批准号: 9978234
• 负责人: Minghao Jin
• 金额: $ 22.05万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978234
• 关键词: 11 cis Retinal; Affect; Age related macular degeneration; All-Trans-Retinol; Apical; Binding; Binding Proteins; Blindness; Cattle; Cell Cycle; Cells; Cellular Retinol Binding Protein; Complex; Defect; Development; Disease; Epithelial Cells; Esterification; Esters; Eye; Family; Functional disorder; G-Protein-Coupled Receptors; Genes; Goals; Homologous Protein; Incubated; Integral Membrane Protein; Knock-out; Knowledge; Leber' s amaurosis; Length; Light; Link; MPP1 gene; Membrane Proteins; Molecular; Molecular Chaperones; Mus; Mutation; Natural regeneration; Neurons; Opsin; Organelles; Patients; Photons; Photoreceptors; Play; Process; Proteins; Research; Research Project Grants; Retina; Retinal Degeneration; Retinal Dystrophy; Retinal Pigments; Retinitis Pigmentosa; Retinoids; Retinol Binding Proteins; Role; Side; Stargardt' s disease; Structure of retinal pigment epithelium; Testing; Visual impairment; Wild Type Mouse; apical membrane; base; cDNA Library; cell type; chromophore; extracellular; in vivo; innovation; interstitial retinol-binding protein; knock-down; lecithin-retinol acyltransferase; membrane-associated guanylate kinase; novel; novel therapeutic intervention; novel therapeutics; photoreceptor degeneration; prevent; prototype; receptor; response; screening; trafficking; uptake; visual cycle

PROJECT SUMMARY/ABSTRACT: The apical membranes of the retinal pigment epithelium (RPE) are associated with the photoreceptor outer segments (POS), the light-sensing organelle of the neurons. POS contain a large amount of the visual pigments consisting of an 11-cis-retinal (11cRAL), the light-sensitive chromophore, and an opsin G protein-coupled receptor. When light hits the visual pigments, photon energy activates opsins by converting 11cRAL to all-trans- retinal (atRAL) in the visual pigments. Since apo-opsins (without 11cRAL) do not respond to light stimulation, 11cRAL must be regenerated and recombined with apo-opsins to form light sensitive visual pigments. It is well established that atRAL produced by the photoisomerization is reduced to all-trans-retinol (atROL) in the POS; atROL is then released from the POS into the interphotoreceptor matrix (IPM) between POS and the RPE apical membranes, and esterified to all-trans retinyl esters (atRE) in RPE in order to synthesize 11cRAL. However, it is largely unknown how RPE uptakes atROL from the IPM and how the esterification of atROL in RPE is regulated. Interphotoreceptor retinoid-binding protein (IRBP) secreted from the photoreceptors is the most abundant soluble protein in the IPM. Mutations in IRBP cause vision impairment and retinal dystrophy in affected patients and Irbp-/- mice. We recently demonstrated that IRBP plays an important role in the retinoid visual cycle. Consistent with these studies, our preliminary studies showed that IRBP promoted intracellular atRE synthesis in the eyecup RPE incubated with the extracellular atROL in the presence of IRBP. This result suggests that IRBP facilitates atROL uptake and/or esterification by RPE. However, the molecular mechanism by which IRBP promotes cellular uptake and esterification of atROL remains completely unknown. The goal of this project is to identify and characterize RPE apical membrane and intracellular proteins involved in the IRBP-dependent atROL uptake and atRE synthesis by RPE. Through screening of bovine RPE cDNA libraries, we have isolated a gene for an intracellular apical protein that promoted atRE synthesis from the extracellular substrate of atROL bound with IRBP. Aim 1 is to elucidate the molecular mechanism by which the apical protein promotes atRE synthesis in RPE. To do this, we will analyze interaction of the protein with cellular retinol-binding protein 1 (CRBP) and/or lecithin:retinol acyltransferase (LRAT) in RPE. Aim 2 is to characterize an apical transmembrane protein as a potential receptor for IRBP. Our preliminary study showed that an RPE apical transmembrane protein interacted with IRBP. We will test if the interaction promotes synthesis of atRE in RPE. We will then test if eliminating the membrane protein in the mouse RPE reduces atRE synthesis. The results of this project will discover previously unknown players involved in the IRBP-dependent atROL uptake and esterification, establishing a novel regulatory mechanism of the visual cycle. The results may also provide a knowledge basis for the development of a new therapy alleviating vision impairment and retinal degeneration in patients with IRBP mutations.

项目总结/摘要： 视网膜色素上皮（RPE）的顶膜与感光细胞外膜相连， 节段（POS），神经元的感光细胞器。POS含有大量的视色素 由光敏发色团11-顺式-视黄醛（11 cRAL）和视蛋白G蛋白偶联的 受体的当光照射到视色素时，光子能量通过将11 cRAL转化为全反式- 视色素中的视网膜（atRAL）。由于脱辅基视蛋白（不含11 cRAL）对光刺激无反应， 11 cRAL必须再生并与脱辅基视蛋白重组以形成光敏视色素。公 确定由光异构化产生的atRAL在POS中还原为全反式视黄醇（atROL）; 然后atROL从POS释放到POS和RPE顶端之间的感光细胞间基质（IPM 膜，并在RPE中酯化成全反式视黄酯（atRE）以合成11 cRAL。但 RPE如何从IPM中摄取atROL以及RPE中atROL的酯化是如何被调节的在很大程度上是未知的。 光感受器分泌的光感受器间维甲酸结合蛋白（IRBP）是最丰富的 IPM中的可溶性蛋白。IRBP突变导致患者视力受损和视网膜营养不良 和Irbp-/-小鼠。我们最近证明，IRBP在类维生素A视觉周期中起着重要作用。 与这些研究一致，我们的初步研究表明IRBP促进细胞内atRE合成 在IRBP存在下与细胞外atROL孵育的眼杯RPE中。该结果表明 IRBP促进RPE对atROL的摄取和/或酯化。然而，IRBP的分子机制 促进atROL的细胞摄取和酯化仍然完全未知。该项目的目标是 鉴定和表征参与IRBP依赖性atROL的RPE顶端膜和细胞内蛋白 通过RPE摄取和atRE合成。通过对牛RPE cDNA文库的筛选，我们分离到一个基因， 对于促进atRE从atROL结合的细胞外底物合成的细胞内顶端蛋白， 关于IRBP目的1阐明顶端蛋白促进atRE合成的分子机制 在RPE中。为此，我们将分析蛋白质与细胞视黄醇结合蛋白1（CRBP）和/或 卵磷脂：视黄醇酰基转移酶（LRAT）。目的2是表征顶端跨膜蛋白作为一个跨膜蛋白。 IRBP的潜在受体。我们的初步研究表明，RPE顶端跨膜蛋白相互作用， 与IRBP。我们将测试相互作用是否促进RPE中atRE的合成。然后我们将测试是否消除 小鼠RPE中的膜蛋白减少atRE合成。该项目的结果将发现以前 参与IRBP依赖性atROL摄取和酯化的未知参与者，建立了一种新的 视觉周期的调节机制。研究结果也可为今后的发展提供知识基础 一种新的治疗方法，减轻IRBP突变患者的视力障碍和视网膜变性。