Biochemical and Genetic Analysis of the Visual Cycle

光感知中的第一个事件是视蛋白色素分子吸收光子，导致11-顺式视黄醛发色团异构化。在恢复光敏性之前，所得到的全反式视黄醛必须通过一种称为视觉循环的酶促过程化学地重新异构化为11-顺式视黄醛。视觉循环的大多数步骤发生在视网膜色素上皮（RPE）内，这是一层与光感受器外节相邻的细胞。视觉循环的一些酶尚未被很好地表征，它们的基因尚未克隆。其中一种酶是异构水解酶，它催化全反式到11顺式的关键再异构化步骤。另一种是全反式视黄醇酯水解酶，它水解所有反式视黄醇酯，生成全反式视黄醇和一种脂肪酸。还有一种是11-顺式视黄酰基酯水解酶，它同样能水解11-顺式视黄酰基酯。虽然这些水解酶的活性已经在RPE膜中得到了很好的记录，但全反式视黄酰基酯水解酶和11-顺式视黄酰基酯水解酶在视觉循环中的作用还不清楚。本方案的第一个具体目的是纯化异构体水解酶、全反式视黄醇酯水解酶和11-顺式视黄醇酯水解酶。这些酶的mRNA和基因将使用两种互补的策略进行克隆。每种蛋白在视觉周期中的功能将通过产生基因零突变的“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，将利用转基因和基因敲除小鼠研究其生化机制。实际上，我们对视觉周期的调节一无所知。我们实验室最近对rpe65-/-敲除小鼠和培养的RPE细胞的两项观察显示光介导的视黄酰基酯动员。这些观察结果表明，RPE细胞本质上对光敏感。此外，这些观察结果表明RPE细胞中存在一种新的信号通路，可以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节途径的光受体分子。本提案的具体目标II和III是利用组织培养和小鼠遗传模型，从生物化学和遗传学上表征这一调控途径。本节的目的是确定这种赋予光敏性的调节途径中的视蛋白光色素，定义G蛋白α亚基，并确定由该途径调节的视觉循环中的催化步骤。

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.