Biochemical and Genetic Analysis of the Visual Cycle

光感知的第一个事件是视蛋白色素分子吸收光子，这会导致11-顺式视黄醛发色团的异构化。在恢复光敏感度之前，产生的全反式视黄醛必须通过一种称为视觉循环的酶过程重新异构化为11-顺式视黄醛。视觉周期的大多数步骤发生在视网膜色素上皮(RPE)内，这是一层毗邻光感受器外节的细胞层。视觉周期中的几种酶还没有被很好地描述，它们的基因也还没有被克隆。一种这样的酶是异位水解酶，它催化关键的全反式到11-顺式再异构化步骤。另一种是全反式视黄酸酯水解酶，它能将所有反式视黄酸酯水解制得全反式视黄醇和一种脂肪酸。还有一种是11-顺式视黄酸酯水解酶，它同样能降解11-顺式视黄酸酯。尽管这些水解酶在RPE膜中的活性已经被很好地记录下来，但全反式视黄酸酯水解酶和11-顺式视黄酸酯水解酶在视觉周期中的功能还不是很清楚。本方案的第一个具体目标是纯化异麦芽水解酶、全反式视黄酸酯水解酶和11-顺式视黄酸酯水解酶。这些酶的mRNA和基因将使用两种互补的策略进行克隆。每种蛋白质在视觉周期中的功能将通过产生该基因零突变的“敲除”小鼠来研究。此外，每种蛋白质的基因都将被评估为人类遗传性视网膜营养不良的原因之一。如果发现致病等位基因，将使用转基因和基因敲除小鼠来研究生化机制。人们对视觉周期的调节几乎一无所知。我们实验室最近在RPE65/-基因敲除小鼠和培养的RPE细胞上观察到的两个结果表明，光介导的视黄酸酯的动员。这些观察表明，RPE细胞对光是天生敏感的。此外，这些观察结果暗示在RPE细胞中存在一种新的信号通路，它调节视觉周期中的一个或多个步骤。此外，我们还初步确定了这一调控途径的光受体分子。这项提案的具体目标II和III是利用组织培养和小鼠遗传模型从生化和遗传学的角度表征这一调控途径。本部分的目的是确定这一提供光敏反应的调节途径的视蛋白光色素，定义G蛋白α亚基，并确定该途径调节的视觉周期中的催化步骤(S)。

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.