IRBP--STRUCTURE AND FUNCTION

维生素A和脂肪酸在视网膜色素变性之间的转运 视网膜上皮(RPE)和神经视网膜是光感受器的关键 结构、功能和发展。光感受器间类维甲酸结合 蛋白质(IRBP)与视黄醇有两个结合位点，并以非共价方式携带 4脂肪酸当量。IRBP似乎将视黄醇在 RPE和光感受器在维生素A循环中的作用，但它在脂肪中的作用 酸的传输还没有被研究过。为了更好地理解 IRBP在疏水分子的运输中，我们将：1)定义其 约束的结构要求，以及2)确定其在 必需脂肪酸二十二碳六烯酸(22：6n-3)的转运 它在杆状外节(ROS)中大量富含。 IRBP由四重重复结构组成。我们的工作假说 是每个重复结合一个脂肪酸当量，但只有两个重复 可以结合一种视黄醇分子。四重重复结构可以具有 在一定程度上是通过基因复制事件在 鱼和两栖动物。我们预计IRBP将发挥重要作用 在22：6N-3的运输中。最后，在每个重复序列中保守的精氨酸 可能为稳定羧基贡献了它们的三角洲-胍基团 在疏水摇篮中含有部分脂肪酸和维甲酸。 我们将在大肠杆菌中表达IRBP的单个重复序列，以定位 并建立结合的化学计量学和特异性。 我们将确定IRBP是否对22：6N-3和运输具有选择性 这种脂肪酸对：1)发育中的视网膜；2)对 ROS盘脱落后RPE的光感受器。我们会找到 功能重要的结构域，通过识别保守的氨基酸 在七鳃鳗、金鱼、非洲爪哇和哺乳动物IRBPs之间。我们将使用聚合酶链式反应 辅助定点突变研究保守的精氨酸的作用 结合脂肪酸和维甲酸。 我们的长期目标是通过定义IRBP的 疏水配体结合的结构要求及其揭示 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.