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

项目摘要 光受体-类视黄酸结合蛋白(IRBP)是由视网膜表达的，它的缺失导致 转基因小鼠的光感受器变性。作为光感受器的主要可溶性成分 基质(IPM)，可接触到视锥和视杆外段、顶端RPE表面和M？ler细胞绒毛。 IRBP保护类维A酸异构体和氧化降解的复杂作用机制 在视觉周期中，靶向它们在上述细胞之间的传递/释放还知之甚少。X射线 IRBP的四个“模块”之一的晶体结构显示了两个疏水的配体结合域。各别 保守的表面带电结构域可以与细胞表面或基质的成分结合。 这一领域的进展一直受到阻碍，因为缺乏一种系统，可以从 从分子到细胞生理层面。在这里，我们利用非洲爪哇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发现导致了我们的假设，即IRBP保护视黄醇免受 在特殊的“疏水空腔”内氧化，在空腔内的结合受变构调节 分离保守的表面带电结构域和脂肪酸结合结构域。将对这一假设进行评估 通过三个相辅相成的具体目标： 目的1.确定IRBP介导的视黄醇从外节清除的机制。 我们假设IRBP通过受体相互作用来清除外节中的视黄醇。这将是 通过监测从外节中去除全反式视黄醇的生理研究来解决， 而旨在揭示视网膜中IRBP结合伙伴的研究。我们预计Asp1081Asn 人类RRP中的突变破坏了这种相互作用。 目的2.确定IRBP在视网膜中是否具有抗氧化剂的功能。我们预计IRBP可以延缓 视杆外节段脂质过氧化，并保护光感受器内维甲酸的氧化状态 矩阵。目标将是确定这一活动的机制。 目的3.确定全长非洲爪哇IRBP的X射线晶体结构。这项研究将导致 全息和脱脂IRBP结构的X-射线晶体结构。我们预计All-Trans和11- 顺式视黄醇结合在一个特殊的疏水空腔内。我们预测配体与该位点的结合是变构的。 受BBA-折叠(浅裂隙)中脂肪酸结合的调节，最终破坏了高度 保守的Asp和Arg残基对附近的视黄醇结合部位有结构性影响。

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.