VISUAL CYCLE PROTEINS

这一提议的假设是CRALBP发挥着基础性作用 视网膜和视网膜色素上皮中的维生素A代谢 (RPE)。该提案统一的长期目标是建立 了解正常结构、功能和功能的分子基础 规范CRALBP，使与CRALBP相关的问题 视力障碍是可以解决的。具体目标集中在两个方面 CRALBP蛋白和基因，包括：定位维甲酸结合口袋 通过测定维甲酸结合特性和配基对rCRALBP的影响 突变蛋白和野生型蛋白的相关构象变化； 寻求CRALBP和RPE 11之间的直接蛋白质-蛋白质相互作用 顺式视黄醇脱氢酶；开发体内系统 表征组织特异性CRALBP基因的表达；以及表征 关于视网膜维生素A代谢的CRALBP基因敲除小鼠， 电生理学和形态学。方法将包括重组 使用细菌和杆状病毒表达系统生产蛋白质， 紫外可见吸收、荧光法分析类维甲酸结合 滴定和~(13)C、~(19)F核磁共振。蛋白质之间的相互作用将是 通过层析、电泳法和免疫学方法寻找和 以电喷雾质谱、序列分析和 由酶/底物载体活性决定。标准分子生物学 将使用制备和测试野生型和PCE-1突变体的方法 在转基因小鼠中构建CRALBP报告基因。维甲酸高效液相色谱， 将使用西方分析、ERG和超结构方法来 CRALBP基因敲除小鼠的特征。人类CRALBP的突变 破坏维甲酸结合与常染色体隐性遗传有关 视网膜色素变性。拟议的研究将导致更好的 了解CRALBP和CRALBP的结构、功能和调控 与AT有关的视觉障碍。

The hypothesis of this proposal is that CRALBP plays a fundamental role in vitamin A metabolism in the retina and retinal pigment epithelium (RPE). The unifying long range goals of the proposal are to establish a molecular basis for understanding the normal structure, function and regulation of CRALBP in order that questions concerning CRALBP-related visual disorders can be answered. Specific aims focus on both the CRALBP protein and gene and include: Mapping the retinoid-binding pocket of rCRALBP by measuring retinoid binding properties and ligand associated conformational changes in mutant and wildtype proteins; Seeking direct protein-protein interaction between CRALBP and RPE 11- cis-retinol dehydrogenase; Developing an in vivo system for characterizing tissue-specific CRALBP gene expression; and characterize CRALBP knockout mice with regard to retinal vitamin A metabolism, electrophysiology and morphology. Methods will include recombinant protein production using bacterial and baculovirus expression systems, retinoid-binding analysis by UV-visible absorbance, fluorescence titrations and 13C and 19F NMR. Protein -protein interactions will be sought by chromatographic, electrophoretic and immunological methods and characterized by electrospray mass spectrometry, sequence analysis and by enzyme/substrate carrier activity. Standard molecular biology methods will be used to prepare and test mouse wildtype and PCE-1 mutant CRALBP reporter gene constructs in transgenic mice. Retinoid HPLC, Western analysis, ERG and ultra structural methods will be used to characterize CRALBP knockout mice. Mutations in human CRALBP that destroy retinoid binding have been linked to autosomal recessive retinitis pigmentosa. The proposed studies will lead to a better understanding of the structure, function and regulation of CRALBP and the visual disorders with which at may be associated.