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

项目摘要 光受体间类维生素A结合蛋白（IRBP）由视网膜表达，它的缺失导致 转基因小鼠的光感受器退化。作为感光细胞间的主要可溶性成分 基质（IPM），可进入视锥细胞和视杆细胞外节、顶端RPE表面和M？ller细胞绒毛， IRBP在保护类维生素A免受异构体和氧化降解， 在视觉周期期间，靶向它们在上述细胞之间的递送/释放知之甚少。x射线 IRBP的四个“模块”之一的晶体结构显示两个疏水配体结合域。单独 保守的表面带电结构域可以起到与细胞表面或基质的组分结合的作用。 这一领域的进展一直受到阻碍，因为缺乏一个系统，可以让研究IRPB的功能， 从分子水平到细胞生理水平。在这里，我们利用Xenopus作为一个系统来理解 其复杂功能所需的结构、运输和细胞相互作用。 我们对文献的回顾和研究结果得出了我们的假设，即IRBP可以保护视黄醇免受 氧化在一个专门的“疏水腔”，并结合在腔是变构调节， 分离的保守表面电荷和脂肪酸结合结构域。将对这一假设进行评估 通过三个互补的具体目标： 目标1.确定IRBP介导的视黄醇从外节清除的机制。 我们推测IRBP通过受体相互作用，从外节清除视黄醇。这将是 通过监测从外节去除全反式视黄醇的生理学研究来解决， 以及旨在揭示视网膜中IRBP结合伴侣的研究。我们预计Asp 1081 Asn 人rRP中的突变体破坏了这种相互作用。 目标2.以确定IRBP是否在视网膜中起抗氧化剂的作用。我们预计IRBP可以延缓 视杆细胞外节脂质过氧化，并保持视色素在感光细胞间的氧化状态 矩阵一个目标是确定这一活动的机制。 目标3.测定非洲爪蟾IRBP全长的X射线晶体结构。这项研究将导致 全息和apo-IRBP结构的X射线晶体结构。我们预计全反式和11- 顺式视黄醇结合在专门的疏水腔内。我们预测，配体结合的网站是变构 调节的脂肪酸结合在bba-折叠（浅裂），最后破坏盐桥之间的高度 保守的Asp和Arg残基对附近的视黄醇结合位点具有结构影响。

Project Summary 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.