IRBP--STRUCTURE AND FUNCTION

IRBP--结构和功能

• 批准号: 3465902
• 负责人: FEDERICO GONZALEZ-FERNANDEZ
• 金额: $ 9.83万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3465902
• 关键词: Agnatha; Rana; Xenopus; biochemical evolution; biological transport; fatty acid transport; goldfish; laboratory rat; polymerase chain reaction; protein structure function; receptor binding; retina; retinal pigment epithelium; retinoid binding proteins; rod cell; site directed mutagenesis; visual photoreceptor

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.

维生素A和脂肪酸在视网膜色素之间的运输 上皮 (RPE) 和神经视网膜对于感光器至关重要 结构、功能和发展。 光感受器间视黄醇结合 蛋白 (IRBP) 有 2 个视黄醇结合位点，并带有非共价键 4 脂肪酸当量。 IRBP 似乎在 RPE 和光感受器在维生素 A 循环中，但其在脂肪中的作用 尚未研究酸转运。 为了理解的作用 IRBP 在疏水性分子的运输中，我们将： 1) 定义它 结合的结构要求，2) 确定其在 二十二碳六烯酸 (22:6n-3)（一种必需脂肪酸）的运输 它在杆外节（ROS）中大大丰富。 IRBP 由四重重复结构组成。 我们的工作假设 是每个重复结合一个脂肪酸当量，但只有2个重复 可以结合视黄醇分子。 四重重复结构可以具有 部分是通过出现之间的基因复制事件产生的 鱼类和两栖动物。 我们预计 IRBP 将发挥重要作用 在运输 22:6n-3 中。 最后，每次重复中保守的精氨酸 可以贡献其δ-胍基团来稳定羧基 疏水摇篮内的脂肪酸和视黄酸部分。 我们将在大肠杆菌中表达 IRBP 的单个重复序列以定位 结合位点并建立结合的化学计量和特异性。 我们将确定 IRBP 对 22:6n-3 是否具有选择性并转运 这种脂肪酸：1) 发育中的视网膜，2) ROS 盘脱落后 RPE 上的光感受器。 我们将定位 通过识别保守的氨基酸来识别功能重要的结构域 七鳃鳗、金鱼、非洲爪蟾和哺乳动物 IRBP 之间。 我们将使用PCR 辅助定点诱变研究保守精氨酸的作用 结合脂肪酸和视黄酸。 我们的长期目标是通过定义 IRBP 来了解 IRBP 的功能 疏水性配体结合和揭示其结构的要求 在脂肪酸运输中的生理作用。