IRBP--STRUCTURE AND FUNCTION

IRBP--结构和功能

• 批准号: 2163046
• 负责人: FEDERICO GONZALEZ-FERNANDEZ
• 金额: $ 10.71万
• 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/2163046
• 关键词: Agnatha; Rana; Xenopus; alternatives to animals in research; 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