IRBP--STRUCTURE AND FUNCTION

维生素A和脂肪酸在视网膜色素层间的转运 视网膜上皮细胞（RPE）和神经视网膜是感光细胞的关键 结构、功能和发展。 光感受器间类维生素A结合 蛋白（IRBP）具有2个视黄醇结合位点， 4脂肪酸当量。 IRBP似乎可以在视网膜之间转运视黄醇。 维生素A循环中的RPE和光感受器，但其在脂肪代谢中的作用 尚未研究酸的转运。 为了理解 IRBP在疏水分子的运输中，我们将：1）定义其 约束力的结构要求，2）确定其在 二十二碳六烯酸（22：6 n-3）的转运，一种必需脂肪酸 其在视杆外节（ROS）中极大地富集。 IRBP由四重重复结构组成。 我们的工作假设 每个重复序列结合一个脂肪酸当量，但只有2个重复序列 可以结合一个视黄醇分子。 四重重复结构可能具有 部分原因是由于出现了基因复制事件， 鱼类和两栖动物。 我们预计IRBP将发挥重要作用 在运输22：6 n-3。 最后，每个重复序列中的保守序列 可以贡献它们的δ-胍鎓基团以稳定羧基 脂肪酸和视黄酸的部分在疏水的摇篮内。 我们将IRBP的单个重复序列在E.大肠杆菌定位 结合位点并建立结合的化学计量和特异性。 我们将确定IRBP是否对22：6 n-3和转运具有选择性 这种脂肪酸：1）发育中的视网膜，2） 在ROS盘脱落后，从RPE释放光感受器。 我们会找到 功能上重要的结构域，通过鉴定保守的氨基酸 七鳃鳗、金鱼、非洲爪蟾和哺乳动物IRBP之间的联系。 我们将使用PCR 辅助定点突变以研究保守氨基酸的作用 结合脂肪酸和视黄酸。 我们的长期目标是通过定义IRBP的功能来了解IRBP的功能。 疏水配体结合和揭示其结构要求 在脂肪酸运输中的生理作用。

The transport of vitamin A and fatty acids between the retinal pigmented epithelium (RPE) and neural retina is critical to photoreceptor structure, function and development. Interphotoreceptor retinoid-binding protein (IRBP) has 2 binding sites for retinol and carries non-covalently 4 fatty acid equivalents. IRBP appears to transport retinol between the RPE and photoreceptors during the vitamin A cycle, but its role in fatty acid transport has not been studied. In order to understand the role of IRBP in the trafficking of hydrophobic molecules, we will: 1) define its structural requirements for binding, and 2) determine its role in the transport of docosahexaenoic acid (22:6n-3), an essential fatty acid which is greatly enriched in the rod outer segments (ROS). IRBP consists of a four-fold repeat structure. Our working hypothesis is that each repeat binds one fatty acid equivalent, but only 2 repeats can bind a molecule of retinol. The four-fold repeat structure may have arisen in part through a genetic duplication event between the emergence of fish and amphibians. We anticipate that IRBP plays an important role in the transport 22:6n-3. Finally, conserved arginines in each repeat may contribute their delta-guanidinium groups to stabilize the carboxyl moiety of fatty and retinoic acids within a hydrophobic cradle. We will express IRBP's individual repeats in E. coli to localize the binding sites and establish the stoichiometry and specificity of binding. We will determine whether IRBP is selective for 22:6n-3 and transports this fatty acid to: 1) the developing retina, and 2) to the photoreceptors from the RPE following ROS disk shedding. We will locate functionally important domains, by identifying amino acids conserved between lamprey, goldfish, Xenopus and mammalian IRBPs. We will use PCR aided site directed mutagenesis to study the role of conserved arginines in binding fatty and retinoic acids. Our long term goal is to understand the function of IRBP by defining its structural requirements for hydrophobic ligand-binding and uncovering its physiological role in fatty acid transport.