SYNTHETIC RETINAL PIGMENTS AND BINDING PROTEINS

描述(申请人提供)：维生素A、视网膜、 是视杆细胞和视锥细胞视觉色素的配基。这 配体可以充当反向激动剂，将蛋白质放入其 非活性构象，或激动剂，将蛋白质移动到活性的 构象和激活视觉传导过程。这么小 因此，分子对视觉过程至关重要。我们的研究主要集中在 该配体与各种杆状和锥状视黄素相互作用的研究 以及一种名为RPE65的蛋白质的作用，它对产生 视网膜的一种形式，与这些视蛋白结合，即11-顺式异构体。类似物 视网膜将与视黄素结合，并可用来操纵这些G蛋白 感受器。利用各种生理学技术，我们建议使用 视网膜类似物解决了一些特别关注的假说 火蜥蜴绿色视杆和三个视锥的研究。有了这些 视网膜的结构修饰形式，蛋白质构象可以改变 使得配基结合或蛋白质活化中的特定步骤可以 接受检查。通过这些研究，我们正在解决这样的假说： 视锥蛋白与11-顺式视网膜有非常不同的相互作用， 它们释放最终产物全反式视黄醇的机制也 有所不同。 RPE65蛋白对11顺式视网膜的产生是至关重要的，尽管 这种蛋白质的确切功能仍不清楚。我们正在测试 假设蛋白质是支架蛋白，参与复合体 这会导致全反式视黄醇的异构化/氧化 11顺式视网膜。使用细胞系HEK296，其本身含有RPE65，以及 视蛋白表达系统为11顺式视网膜提供了一个陷阱，我们 建议确定哪些其他维甲酸结合/代谢蛋白是 对这一过程至关重要。其次，使用动物模型，这些动物模型有一个或多个 更多的维甲酸结合蛋白被敲除，我们将检测其水平 RPE65和动物产生11顺式视网膜的能力。 现在清楚地表明，许多视网膜疾病是由于在 视黄酸代谢或配体与视觉色素的结合。示例 有多种：Stargardt病，常染色体隐性遗传性视网膜色素变性，以及 莱伯氏先天性黑猩猩。该项目的重点是确定 视黄醇代谢和视黄醇蛋白相互作用的机制 对这些失明疾病的预防或治疗的合理设计 是可以承担的。

DESCRIPTION (provided by applicant): The aldehyde form of vitamin A, retinal, is the ligand of the visual pigments in both rod and cone photoreceptors. This ligand can either act as an inverse agonist, placing the protein into its inactive conformation, or an agonist, moving the protein into an active conformation and activating the visual transduction process. This small molecule is thus critical to the visual process. Our research is focused on the study of the interaction of this ligand with the various rod and cone opsins and the role of one protein, RPE65, which is critical for the generation of the form of retinal that binds to those opsins, the 11-cis isomer. Analogues of retinal will bind to the opsins and can be used to manipulate these G-protein receptors. Using a variety of physiological techniques, we propose to use retinal analogues to address a number of hypotheses particularly focused on studies of the green rod and three cone opsins of the salamander. With these structurally modified forms of retinal, the protein conformation can be altered such that particular steps in the ligand binding or the protein activation can be examined. Through these studies, we are addressing hypotheses that the rod and cone opsins have very different interactions with 11-cis-retinal and that their mechanisms for the release of the end product, all-trans-retinol, also differs. The protein RPE65 is critical for the generation of 11-cis-retinal, although the exact function of this protein is still unknown. We are testing the hypothesis that the protein is a scaffolding protein, involved in a complex that brings about the isomerization/oxidation of all-trans-retinol to 11-cis-retinal. Using a cell line HEK296, which itself contains RPE65, and an opsin expression system which then provides a "trap" for the 11-cis-retinal, we propose to determine which other retinoid-binding/metabolizing proteins are critical for this process. Secondly, using animal models, which have had one or more of the retinoid-binding proteins knocked out, we will examine the levels of RPE65 and the ability of the animal to produce 11-cis-retinal. It is now being clearly shown that many retinal disorders are due to errors in retinoid metabolism or binding of the ligand with the visual pigments. Examples are various: Stargardt's disease, autosomal recessive retinitis pigmentosa, and Leber's congenital amaurosis. This project is focused on determining the mechanisms of retinoid metabolism and retinal-opsin protein interactions so that a rational design of prevention or therapies for these blinding disorders can be undertaken.