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: 10547900
• 负责人: Jian-Xing Ma
• 金额: $ 31.66万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 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 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10547900
• 关键词: Studies; retinyl; ester; hydrolase; visual

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.

项目总结/摘要 光诱导视色素发色团11-顺式视黄醛异构化为全反式视黄醛 启动视觉。11-顺式视黄醇通过视觉循环的有效再循环对于视觉系统的形成是必不可少的。 色素和维持正常视力。视觉循环的一个关键步骤是全反式视黄酯的转化 在视黄醇异构酶RPE 65的催化下，转化为11-顺式视黄醇。文献研究表明，类维生素A 大部分储存为全反式视黄酯，异构酶RPE 65的底物，在脂滴（视网膜小体）中， 的RPE。然而，RPE 65位于ER中，而不是靠近脂滴。目前尚不清楚其疏水性 RPE 65的底物全反式视黄酯从脂滴转运到ER。这意味着 视觉周期中的重要知识差距。视黄基酯水解酶（REH）可以存在于脂滴中， 动员全反式视黄酯以提供ER中RPE 65异构酶的底物。类马铃薯 含有磷脂酶结构域的蛋白2（PNPLA 2）被称为具有REH的脂肪甘油三酯脂肪酶 在肝脏中的活动。其在RPE中的功能及其与视觉周期的关联尚未研究。 我们的初步研究在RPE和脂滴中鉴定了PNPLA 2。重要的是，PNPLA 2-/-小鼠显示 降低ERG反应和延迟视色素再生。PNPLA 2 KO也导致降低的 11-顺式视网膜生成和全反式视黄酯在RPE中的水平增加，这表明视网膜色素上皮细胞中的全反式视黄酯水平降低。 异构酶活性这些发现表明PNPLA 2在视觉周期中的潜在作用。我们假设 PNPLA 2在RPE中起REH的作用，并从脂滴中动员全反式视黄酯，以提供 ER中RPE 65异构酶产生11-顺式视黄醇的足够底物。在这个项目中，我们将定义 PNPLA 2在维持视觉周期和正常视力中的作用。我们将使用RPE特定的PNPLA 2 条件性KO小鼠以研究PNPLA 2 KO对视网膜功能、视网膜变性、视色素 11-顺式视黄醛的形成和再生。我们还计划确定PNPLA 2与 RPE 65基因转染RPE 65-/-小鼠视网膜色素上皮细胞后，可增强RPE 65对视力恢复的作用 功能，视色素形成和11-顺式视网膜再生，与单独的RPE 65基因递送相比。我们 还将阐明PNPLA 2促进RPE 65异构酶活性和加速RPE 65异构酶活性的机制。 11-顺式视网膜再生在视觉周期。我们将研究在RPE细胞中敲除PNPLA 2是否会 PNPLA 2的过表达可促进RPE 65异构酶活性，减少脂滴 在RPE细胞中。我们还将确定PNPLA 2激活剂是否会促进，而PNPLA 2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组成部分 并解决视力方面的一个重要知识缺口。该项目还将确定PNPLA 2的新功能， 的RPE。PNPLA 2有可能改善RPE 65基因治疗由RPE 65引起的视网膜营养不良 突变。