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

描述（由申请人提供）： 光感受器间视色素结合蛋白（IRBP）由视网膜表达，其缺失导致转基因小鼠的光感受器变性。作为感光细胞间基质（interphotoreceptor matrix，IPM）的主要可溶性成分，它可以进入视锥细胞和视杆细胞的外节、顶端RPE表面，|IRBP在保护类维生素A免受异构和氧化降解同时靶向其在视觉周期期间在上述细胞之间的递送/释放的复杂功能的机制知之甚少。IRBP的四个“模块”之一的X射线晶体结构显示两个疏水配体结合域。单独的保守表面带电结构域可以起到结合细胞表面或基质的组分的作用。由于缺乏一个从分子水平到细胞生理水平研究IRPB功能的系统，阻碍了该领域的进展。在这里，我们利用非洲爪蟾作为一个系统来了解其复杂功能所需的结构，贩运和细胞相互作用。我们对文献的回顾和研究结果导致我们的假设，IRBP保护视黄醇在一个专门的“疏水腔”内免受氧化，并且腔中的结合是由单独的保守表面电荷和脂肪酸结合结构域变构调节的。这一假设将通过3个互补的具体目标进行评估：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。这将通过监测从外节去除全反式视黄醇的生理学研究以及旨在揭示视网膜中IRBP结合伴侣的研究来解决。我们预计，Asp 1081 Asn突变体在人类rRP破坏这种相互作用。目标2.以确定IRBP是否在视网膜中起抗氧化剂的作用。我们预期IRBP可以延缓视杆细胞外段脂质过氧化，并保护视黄酸在感光细胞间基质中的氧化状态。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-顺式视黄醇结合在一个专门的疏水腔。我们预测，配体结合的网站是变构调节的脂肪酸结合的bba-折叠（浅裂），最后破坏的盐桥之间的高度保守的天冬氨酸和精氨酸残基的结构后果，以附近的视黄醇结合位点。 公共卫生相关性： 光感受器间视色素结合蛋白（IRBP）是影响视网膜的疾病的候选者。缺乏这种蛋白质会导致小鼠感光细胞死亡，而这种蛋白质的突变会导致视网膜色素变性。IRBP完成了在光感受器和附近细胞之间运输不同化学形式的维生素A的出色工作。同时，IRBP保护维生素A免受氧化损伤。我们的研究旨在揭示IRBP如何做到这一点，以便我们能够了解并最终治疗涉及维生素A循环的疾病。在这项研究中，IRBP的贩运，生物化学和3-D结构的非洲爪蟾，非洲爪蛙，其大型和可访问的光感受器促进蛋白质贩运和功能的研究。

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.