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.

项目摘要/摘要 光诱导的11-cis视网膜的异构化，视觉色素的发色团，全型视网膜 启动愿景。通过视觉周期对11盘视网膜的有效回收，对于形成视觉至关重要 色素并保持正常视力。视觉周期的关键步骤是全元视网膜酯的转化 到11-CIS视黄醇，由视黄醇异构酶RPE65催化。有记录的研究表明，类视黄素是 在脂质液滴（视网膜体）中，在很大程度上存储为全反式视网膜酯，是异构酶RPE65的底物， RPE。但是，RPE65位于ER中，而不是脂质液滴附近。未知如何疏水 RPE65的底物全反式视网膜酯从脂质液滴转移到ER。这代表一个 视觉周期中的重要知识差距。脂质液滴中可能存在视黄酯水解酶（REH） 动员全反式视网膜酯为ER中的RPE65异构酶提供底物。类似patatine 含有蛋白2（PNPLA2）的磷脂酶结构域被称为脂肪甘油三酸酯脂肪酶，具有REH 肝脏的活性。尚未研究其在RPE中的功能及其与视觉周期的关联。 我们的初步研究确定了RPE和脂质液滴中的PNPLA2。重要的是，PNPLA2 - / - 小鼠显示 ERG反应下降和视觉色素再生的延迟。 PNPLA2 KO也导致减少 RPE中的11-CIS视网膜产生和全反式视网膜酯的水平增加，表明降低 异构酶活性。这些发现表明PNPLA2在视觉周期中的潜在作用。我们假设这一点 PNPLA2在RPE中充当REH，并从脂质液滴中动员全元视网膜酯以提供 ER中RPE65异构酶的足够底物可产生11-CIS视黄醇。在这个项目中，我们将定义 PNPLA2在保持视觉周期和正常视力中的作用。我们将使用RPE特异性PNPLA2 有条件的KO小鼠研究PNPLA2 KO对视网膜功能，视网膜变性，视觉色素的影响 11盘视网膜的形成和再生。我们还计划确定PNPLA2的共表达以及 rpe65中的RPE65通过基因递送的RPE65 - / - 小鼠将增强RPE65对恢复视觉的影响 与仅RPE65基因递送相比，功能，视觉色素形成和11-CIS视网膜再生。我们 还将阐明PNPLA2促进RPE65异构酶活性并加速的机制 在视觉周期中11盘视网膜再生。我们将研究RPE细胞中PNPLA2的敲除是否会 减少PNPLA2的过表达将促进RPE65的异构酶活性并减少脂质液滴 在RPE细胞中。我们还将确定PNPLA2激活剂是否会促进，而PNPLA2抑制剂将抑制 RPE细胞中的异构酶活性。这些研究有可能在视觉周期中识别缺失的组件 并解决视力中的重要知识差距。该项目还将确定pnpla2的新功能 RPE。 PNPLA2具有改善由RPE65引起的视网膜营养不良的RPE65基因治疗 突变。