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.

项目摘要/摘要： 视网膜色素上皮(RPE)的顶膜与光感受器外层相关 神经节(POS)，神经元的感光细胞器。POS含有大量的视觉色素 由11-顺式视网膜(11-cre)、光敏发色团和视蛋白G蛋白偶联组成 受体。当光线照射到视觉色素时，光子能量通过将11cral转换为全反式光激活视蛋白。 视色素中的视网膜。由于凋亡素(不含11cre)对光刺激没有反应， 11细胞必须再生，并与凋亡素重组，形成光敏的视觉色素。这很好 确定在POS中，光异构化产生的ATAL被还原为全反式视黄醇(AtROL)； 然后atROL从POS释放到POS和RPE根尖之间的光感受器间矩阵(IPM)中 膜，然后在RPE中酯化成全反式视黄酸酯(ATRE)，以合成11cre。然而，它 在很大程度上还不清楚RPE如何从IPM中摄取atROL，以及atROL在RPE中的酯化是如何调节的。 光感受器分泌的视黄醇结合蛋白(IRBP)含量最丰富 IPM中的可溶性蛋白质。IRBP基因突变导致患者视力障碍和视网膜营养不良 和IRBP-/-小鼠。我们最近证明了IRBP在视黄醇的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促进了细胞内atre的合成。 在IRBP存在的情况下，RPE与细胞外atROL孵育。这一结果表明 IRBP促进RPE摄取和/或酯化ROL。然而，IRBP的分子机制 促进细胞摄取和酯化atROL仍是完全未知的。这个项目的目标是 RPE根尖膜和细胞内参与IRBP依赖性atROL的蛋白的鉴定和特性 RPE摄取和atre合成。通过对牛RPE基因文库的筛选，我们分离到一个基因 用于促进atROL结合的胞外底物合成atre的细胞内顶端蛋白 与IRBP合作。目的1阐明顶端蛋白促进atre合成的分子机制。 在RPE中。为此，我们将分析该蛋白质与细胞视黄醇结合蛋白1(CRBP)和/或 卵磷脂：RPE中的视黄醇酰基转移酶(LRAT)。目标2是将顶端跨膜蛋白表征为 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合作。我们将测试这种相互作用是否促进RPE中ATRE的合成。然后我们将测试是否消除了 小鼠视网膜色素上皮细胞膜蛋白减少血管紧张素转换酶的合成。这个项目的结果将会在之前发现 参与IRBP依赖的atROL摄取和酯化的未知参与者，建立了一个新的 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 一种减轻IRBP突变患者视力障碍和视网膜变性的新疗法。