Biochemical and Genetic Analysis of the Visual Cycle

视觉周期的生化和遗传分析

• 批准号: 7121103
• 负责人: GABRIEL H TRAVIS
• 金额: $ 37.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-09 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7121103
• 关键词: RNA interference; affinity chromatography; chromophore; enzyme mechanism; gene expression; gene targeting; genetic models; genetically modified animals; hydrolase; isomer; laboratory mouse; messenger RNA; molecular biology; molecular cloning; photostimulus; protein purification; protein structure function; retinal pigment epithelium; retinaldehyde; rhodopsin; tissue /cell culture; vision; visual perception; visual pigments

The first event in light perception is absorption of a photon by an opsin pigment molecule, which causes isomerization of the 11-cis-retinaldehyde chromophore. Before light sensitivity can be restored, the resulting all-tans-retinaldehyde must be chemically re-isomerized to 11-cis-retinaldehyde by an enzymatic process called the visual cycle. Most steps of the visual cycle take place within the retinal pigment epithelium (RPE), a layer of cells adjacent to the photoreceptor outer-segments. Several enzymes of the visual cycle have not been well characterized and their genes not yet cloned. One such enzyme is isomerohydrolase, which catalyzes the critical all-trans to 11-cis re-isomerization steps. Another is all-trans-retinyl ester hydrolase, which hydrolyzes all trans-retinyl esters to yield all-trans-retinol and a fatty acid. Still another is 11-cis-retinyl ester hydrolase, which similarly hydrolyzes 11-cis-retinyl esters. Although these hydrolase activities have been well documented in RPE membranes, the functions of all-trans-retinyl ester hydrolase and 11-cis-retinyl ester hydrolase in the visual cycle are not well understood. The first Specific Aim of this proposal is to purify isomerohydrolase, all-trans-retinyl ester hydrolase, and 11-cis-retinyl ester hydrolase. The mRNA's and genes for these enzymes will be cloned using two complementary strategies. The function of each protein in the visual cycle will be studied by generating 'knockout' mice with a null mutation in the gene. Also, the gene for each protein will be evaluated as a cause of inherited retinal dystrophy in humans. If disease-causing alleles are found, the biochemical mechanisms will be studied using transgenic and knockout mice. Virtually nothing is known about regulation of the visual cycle. Two recent observations in our laboratory on rpe65-/- knockout mice and cultured RPE cells showed light-mediated mobilization of retinyl esters. These observations indicate that RPE cells are intrinsically sensitive to light. Further, these observations imply the existence of a novel-signaling pathway in RPE cells that regulates one or more steps in the visual cycle. Also, we have tentatively identified the light-receptor molecule for this regulatory pathway. Specific Aims II and III of this proposal are to characterize this regulatory pathway biochemically and genetically, using tissue culture and mouse genetic models. The goals of this section are to identify the opsin photopigment for this regulatory pathway that confers light-sensitivity, to define the G protein alpha-subunit, and to determine the catalytic step(s) in the visual cycle regulated by this pathway.

光感知中的第一个事件是由视蛋白色素分子吸收光子，这导致11-顺式-视黄醇发色团的异构化。在恢复光敏感性之前，所产生的全反式视黄醇必须通过称为视觉循环的酶促过程化学异构化为11-顺式视黄醇。视觉周期的大多数步骤发生在视网膜色素上皮（RPE）内，视网膜色素上皮是与感光器外节相邻的细胞层。视觉周期中的几种酶还没有很好的表征，它们的基因也没有克隆。一种这样的酶是异构水解酶，其催化关键的全反式至11-顺式再异构化步骤。另一种是全反式视黄酯水解酶，其水解所有反式视黄酯以产生全反式视黄醇和脂肪酸。另一种是11-顺式-视黄酯水解酶，其类似地水解11-顺式-视黄酯。虽然这些水解酶的活动已被很好地记录在视网膜色素上皮细胞膜，全反式视黄酯水解酶和11-顺式视黄酯水解酶在视觉周期的功能还没有得到很好的理解。本建议的第一个具体目的是纯化异构水解酶、全反式视黄酯水解酶和11-顺式视黄酯水解酶。将使用两种互补策略克隆这些酶的mRNA和基因。每种蛋白质在视觉周期中的功能将通过产生基因中具有无效突变的“敲除”小鼠来研究。此外，每种蛋白质的基因将被评估为人类遗传性视网膜营养不良的原因。如果发现致病等位基因，将使用转基因和基因敲除小鼠研究其生化机制。事实上，人们对视觉周期的调节一无所知。我们实验室最近对rpe 65-/-敲除小鼠和培养的RPE细胞的两项观察结果表明，光介导的视黄酯动员。这些观察结果表明，RPE细胞本质上对光敏感。此外，这些观察结果意味着在RPE细胞中存在一种新的信号通路，调节视觉周期中的一个或多个步骤。此外，我们已经初步确定了这种调控途径的光受体分子。本提案的具体目标II和III是使用组织培养和小鼠遗传模型对该调控途径进行生化和遗传表征。本节的目的是鉴定赋予光敏感性的调节途径的视蛋白色素，定义G蛋白α亚基，并确定由该途径调节的视觉周期中的催化步骤。