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.

项目概要/摘要 光诱导 11-顺式视黄醛（视色素的发色团）异构化为全反式视黄醛 启动愿景。 11-顺式视网膜通过视觉循环的有效回收对于视觉的形成至关重要 色素并维持正常视力。视觉循环的关键步骤是全反式视黄酯的转化 由视黄醇异构酶 RPE65 催化生成 11-顺式视黄醇。有记录的研究表明，类维生素A 大部分以全反式视黄酯（异构酶 RPE65 的底物）形式储存在脂滴（视黄体）中 RPE。然而，RPE65 位于内质网而不是脂滴附近。疏水性如何尚不清楚 全反式视黄酯是 RPE65 的底物，从脂滴转运至内质网。这代表了一个 视觉循环中的重要知识差距。视黄酯水解酶 (REH) 可能存在于脂滴中，以 动员全反式视黄酯为 ER 中的 RPE65 异构酶提供底物。类铜绿 含有蛋白 2 (PNPLA2) 的磷脂酶结构域被称为具有 REH 的脂肪甘油三酯脂肪酶 肝脏中的活性。其在 RPE 中的功能及其与视觉周期的关联尚未得到研究。 我们的初步研究在 RPE 和脂滴中发现了 PNPLA2。重要的是，PNPLA2-/- 小鼠表现出 ERG 反应下降，视色素再生延迟。 PNPLA2 KO 还导致减少 RPE 中 11-顺式视黄醛生成和全反式视黄酯水平增加，表明 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 PNPLA2 在 RPE 中起到 REH 的作用，并从脂滴中动员全反式视黄酯，以提供 ER 中的 RPE65 异构酶有足够的底物生成 11-顺式视黄醇。在这个项目中，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 RPE 专用的 PNPLA2 条件性敲除小鼠，研究 PNPLA2 敲除对视网膜功能、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 是否与 通过基因导入RPE65-/-小鼠的RPE中的RPE65将增强RPE65恢复视力的效果 与单独的 RPE65 基因递送相比，功能、视觉色素形成和 11-顺式视网膜再生。我们 还将阐明PNPLA2促进RPE65异构酶活性并加速的机制 视觉周期中的 11-顺式视网膜再生。我们将研究在 RPE 细胞中敲除 PNPLA2 是否会 降低，而 PNPLA2 的过度表达会促进 RPE65 异构酶活性并减少脂滴 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。这些研究有可能识别视觉周期中缺失的组成部分 并解决视觉方面的重要知识差距。该项目还将确定 PNPLA2 的一个新功能 RPE。 PNPLA2 有潜力改善 RPE65 基因治疗 RPE65 引起的视网膜营养不良 突变。