Use of Xenopus a system to study the trafficking,function and structure of IRBP

利用Xenopus系统研究IRBP的运输、功能和结构

• 批准号: 7797727
• 负责人: FEDERICO GONZALEZ-FERNANDEZ
• 金额: --
• 依托单位: VA WESTERN NEW YORK HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7797727
• 关键词: 11-cis-Retinol; 3-Dimensional; Address; Affect; African; All-Trans-Retinol; Antioxidants; Apical; Binding; Binding Sites; Biochemistry; Cell Death; Cell surface; Cells; Charge; Chemicals; Cleaved cell; Cloning; Complex; Crystallography; Disease; Escherichia coli; Excision; Fatty Acids; Funding; GRB10 gene; Goals; Human; Individual; Lead; Length; Ligand Binding; Ligand Binding Domain; Lipid Peroxidation; Mediating; Molecular; Monitor; Mus; Mutation; Occupations; Pattern; Photoreceptors; Physiological; Proteins; Research; Retina; Retinal Cone; Retinitis Pigmentosa; Retinoids; Review Literature; Rod Outer Segments; Roentgen Rays; Site; Sodium Chloride; Structure; Surface; System; Time; Transgenic Mice; Villus; Vitamin A; X ray diffraction analysis; X-Ray Diffraction; Xenopus; Xenopus sp.; interstitial retinol-binding protein; mutant; novel; oxidation; oxidative damage; photoreceptor degeneration; protein function; protein structure; protein transport; public health relevance; receptor; scaffold; trafficking; visual cycle

DESCRIPTION (provided by applicant): Interphotoreceptor-retinoid binding protein (IRBP) is expressed by the retina, and its absence leads to photoreceptor degeneration in transgenic mice. As the major soluble component of the interphotoreceptor matrix (IPM), has access to the cone and rod outer segments, apical RPE surface, and M|ller cell villi, The mechanism for IRBP's complex function in protecting retinoids from isomeric and oxidative degradation while targeting their delivery/release between the above cells during the visual cycle is poorly understood. X-ray crystal structure of one of IRBP's four "modules" shows two hydrophobic ligand-binding domains. Separate conserved surface charged domains could function to bind to components of the cell surface or matrix. Progress in this field has been hampered by the lack of a system that could allow study IRPB's function from the molecular to cellular-physiological levels. Here, we take advantage of Xenopus as a system to understand the structure, trafficking and cellular interactions required for its complex function. Our review of the literature, and findings have lead to our Hypothesis that IRBP protects retinol from oxidation within a specialized "hydrophobic cavity", and binding in cavity is allosterically regulated by separate conserved surface charged, and fatty acid binding domains. This hypothesis will be evaluated through 3 complementary specific aims: Aim 1. To determine the mechanism for IRBP mediated clearance of retinol from the outer segments. We hypothesize that IRBP interacts through a receptor to clear retinol from the outer segments. This will be addressed through physiological studies monitoring the removal of all-trans retinol from the outer segments, and studies aimed at uncovering binding partners for IRBP in the retina. We anticipate that the Asp1081Asn mutant in human rRP disrupts this interaction. Aim 2. To determine if IRBP's functions as an antioxidant in the retina. We anticipate that IRBP can retard rod outer segment lipid peroxidation, and preserve the oxidative state of retinoids within the interphotoreceptor matrix. A goal will be to determine the mechanism of this activity. Aim 3. To determine the X-ray crystal structure of full-length Xenopus IRBP. This research will lead to the X-ray crystal structure of both the holo- and apo-IRBP structures. We anticipate that that all-trans and 11- cis retinol bind within a specialized hydrophobic cavity. We predict that ligand binding to the site is allosterically regulated by fatty acid binding in the bba-fold (shallow cleft), Finally disruption of a salt bridge between highly conserved Asp and Arg residues has structural consequences to the nearby retinol binding site. PUBLIC HEALTH RELEVANCE: Interphotoreceptor-retinoid binding protein (IRBP), is a candidate for diseases that affect the retina. Absence of the protein in mice causes photoreceptor cells death, and mutation of the protein causes retinitis pigmentosa. IRBP accomplishes a remarkable job of transporting different chemical forms of vitamin A between the photoreceptors and nearby cells. At the same time, IRBP protects vitamin A from oxidative damage. Our research is directed at uncovering how IRBP does this so that we can understand, and eventually treat, diseases that involve the vitamin A cycle. In this research, the trafficking, biochemistry, and 3-D structure of IRBP is studied in Xenopus, the African clawed frog whose large and accessible photoreceptors facilitate research in protein trafficking and function.

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