Studies of retinyl ester hydrolase in the visual cycle

视黄酯水解酶在视觉循环中的研究

• 批准号: 10278989
• 负责人: Jian-Xing Ma
• 金额: $ 4.59万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2021-12-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10278989
• 关键词: 11 cis Retinal; 11-cis-Retinol; Address; Adenoviruses; Adipose tissue; Age; All-Trans-Retinol; CRISPR/Cas technology; Cell membrane; Cells; Disease; Enzymes; Esters; Gene Delivery; Generations; Genes; Genetic; High Pressure Liquid Chromatography; Hydrophobicity; Isomerase; Knock-out; Knockout Mice; Knowledge; Light; Lipase; Lipids; Liver; Measures; Monitor; Mus; Mutation; Natural regeneration; Oleates; Opsin; Optical Coherence Tomography; Pathway interactions; Phospholipase; Photobleaching; Photons; Photophobia; Photoreceptors; Play; Proteins; RPE65 protein; Recovery; Recycling; Retina; Retinal Degeneration; Retinal Dystrophy; Retinal Pigments; Retinal gene therapy; Retinoids; Rhodopsin; Role; Signal Transduction; Vision; Vitamin A; absorption; chromophore; comparative; conditional knockout; improved; inhibitor/antagonist; lecithin-retinol acyltransferase; lipid transport; overexpression; response; retinal regeneration; retinol isomerase; retinyl esterase; treatment strategy; visual cycle

PROJECT SUMMARY/ABSTRACT Light-induced isomerization of 11-cis retinal, the chromophore of visual pigments, to all-trans retinal initiates vision. Efficient recycling of 11-cis retinal through the visual cycle is essential for the formation of visual pigments and maintaining normal vision. A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cis retinol, catalyzed by RPE65, the retinol isomerase. Documented studies have shown that retinoids are largely stored as all-trans retinyl ester, the substrate of the isomerase RPE65, in lipid droplets (retinosomes) in the RPE. However, RPE65 is located in the ER and not near the lipid droplets. It is unknown how hydrophobic all-trans retinyl ester, the substrate of RPE65, is transported from lipid droplets to the ER. This represents an important knowledge gap in the visual cycle. A retinyl ester hydrolase (REH) may be present in lipid droplets to mobilize all-trans retinyl ester to provide the substrate for the RPE65 isomerase in the ER. Patatine-like phospholipase domain containing protein 2 (PNPLA2) is known as an adipose triglyceride lipase with an REH activity in the liver. Its function in the RPE and its association with the visual cycle have not been investigated. Our preliminary studies identified PNPLA2 in the RPE and in lipid droplets. Importantly, PNPLA2-/- mice showed declined ERG responses and delayed regeneration of visual pigments. PNPLA2 KO also resulted in reduced 11-cis retinal generation and increased levels of all-trans retinyl ester in the RPE, suggesting a decreased isomerase activity. These findings suggest a potential role of PNPLA2 in the visual cycle. We hypothesize that PNPLA2 functions as an REH in the RPE and mobilizes all-trans retinyl ester from lipid droplets to provide sufficient substrate for the RPE65 isomerase in the ER to generate 11-cis retinol. In this project, we will define the role of PNPLA2 in maintaining the visual cycle and normal vision. We will use RPE-specific PNPLA2 conditional KO mice to study the impacts of PNPLA2 KO on retinal function, retinal degeneration, visual pigment formation and regeneration of 11-cis retinal. We also plan to determine if co-expression of PNPLA2 together with RPE65 in the RPE of RPE65-/- mice by gene delivery will enhance the effect of RPE65 on restoring visual function, visual pigment formation and 11-cis retinal regeneration, compared to RPE65 gene delivery alone. We will also elucidate the mechanism by which PNPLA2 promotes the RPE65 isomerase activity and accelerates 11-cis retinal regeneration in the visual cycle. We will investigate if knockout of PNPLA2 in RPE cells will decrease, while overexpression of PNPLA2 will promote, isomerase activity of RPE65 and reduce lipid droplets in RPE cells. We will also determine if a PNPLA2 activator will promote, while a PNPLA2 inhibitor will inhibit the isomerase activity in RPE cells. These studies have potential to identify a missing component in the visual cycle and address an important knowledge gap in vision. This project will also identify a new function of PNPLA2 in the RPE. PNPLA2 has potential to improve the RPE65 gene therapy for retinal dystrophies caused by RPE65 mutations.

项目摘要/摘要 光诱导视觉色素生色团11-顺式视网膜异构化为全反式视网膜 启蒙远见。11顺式视网膜在视觉周期中的有效循环对于视觉的形成至关重要。 色素和维持正常视力。视觉周期中的一个关键步骤是全反式视黄酸酯的转化 由视黄醇异构酶RPE65催化合成11-顺式视黄醇。有记录的研究表明，维甲酸是 主要以全反式视黄酸酯的形式储存在脂滴(视网膜小体)中，RPE65异构酶的底物 RPE。然而，RPE65定位于内质网，而不是靠近脂滴。目前尚不清楚疏水性有多强 全反式视黄酸酯是RPE65的底物，从脂滴运输到内质网。这表示一个 视觉周期中的重要知识缺口。视黄酸酯水解酶(REH)可能存在于脂滴中，以 动员全反式视黄酸酯为内质网中的RPE65异构酶提供底物。帕塔廷式的 磷脂酶结构域含蛋白2(PNPLA2)是一种含REH的脂肪甘油三酯脂肪酶 肝脏的活动。它在RPE中的功能及其与视觉周期的关系还没有被研究过。 我们的初步研究发现，PNPLA2存在于RPE和脂滴中。重要的是，PNPLA2-/-小鼠表现出 ERG反应下降，视觉色素再生延迟。PNPLA2 KO也导致减少 11顺式视网膜生成和RPE中全反式视黄酸酯水平增加，表明 异构酶活性。这些发现提示了PNPLA2在视觉周期中的潜在作用。我们假设 PNPLA2在RPE中作为REH发挥作用，并从脂滴中动员全反式视黄酸酯，以提供 内质网中RPE65异构酶产生11-顺式视黄醇的足够底物。在本项目中，我们将定义 PNPLA2在维持视觉周期和正常视力中的作用。我们将使用特定于RPE的PNPLA2 条件性KO小鼠研究PNPLA2 KO对视网膜功能、视网膜变性、视色素的影响 11顺式视网膜的形成和再生。我们还计划确定PNPLA2与PNPLA2的共同表达 RPE65基因导入RPE65-/-小鼠RPE中的RPE65将增强RPE65恢复视力的作用 功能、视觉色素形成和11顺式视网膜再生，与单独交付RPE65基因相比。我们 还将阐明PNPLA2促进RPE65异构酶活性并加速 11顺式视网膜在视觉周期中的再生。我们将研究在RPE细胞中敲除PNPLA2是否会 PNPLA2过表达会促进RPE65的异构酶活性，减少脂滴 在RPE细胞中。我们还将确定PNPLA2激活剂是否会促进，而PNPLA2抑制剂是否会抑制 RPE细胞中的异构酶活性。这些研究有可能确定视觉周期中缺失的成分 并解决视觉方面的一个重要的知识鸿沟。该项目还将确定PNPLA2在 RPE。PNPLA2有可能改进RPE65对RPE65所致视网膜营养不良的基因治疗 突变。