STUDIES OF VISUAL CYCLE PROTEINS

拟议研究的主要重点是描述 编码细胞视黄醛结合蛋白的人类基因 (CRALBP)，并确定视网膜内的功能结构域 CRALBP的三维结构。CRALBP可能是一个 视觉循环的功能成分，可能起到 底物载体和/或立体选择剂。CRALBP携带AS 内源性配体类维甲酸仅在 视觉，即11-顺式视黄醇和11-顺式视黄醛，只有 在对光有反应的组织(视网膜和松果体)中被发现。 对克隆和测序给予适当的关注 人CRALBP基因的分析与鉴定 负责组织特异性表达的调控区域。一个 还将作出协调一致的努力来确定域 负责视黄醇的结合和与视网膜的相互作用 色素上皮(RPE)视黄醇脱氢酶和视黄酯 合成酶。蛋白质的细胞定位将是 经原位技术证实，该基因的染色体定位 确定了人类基因，并进行了临床连锁研究。这个 CRALBP可能参与遗传性视网膜疾病，如 色素性视网膜炎引起人们的极大关注。远景目标 是通过基因/蛋白质工程研究来定义 与其有关的蛋白质的结构特征 高立体选择性和低感光性。 基因的调节区将由初级RPE来表征 和Muller细胞培养、转染CAT实验。CRALBP基因 将在RPE和Muller细胞培养中测量以下水平 使用各种类固醇激素、生长因子和 维甲酸。整个蛋白质将在化学合成过程中 通过自动控制多肽数量(16-30个残基/多肽) 固相法和抗CRALBP多肽抗体 兔子。功能域将通过筛选 功能阻断抗体和多肽及其限制性 蛋白质分解。拟议研究的综合结果将 提供了一个精确的分子框架，其中涉及的问题 CRALBP的正常功能及其可能的视力障碍 可以用特定的术语回答与关联。

The major emphasis of the proposed research is to characterize the human gene encoding the cellular retinaldehyde-binding protein (CRALBP) from retina and to identify functional domains within the three-dimensional structure of CRALBP. CRALBP could be a functional component of the visual cycle, perhaps serving as a substrate carrier and/or stereoselective agent. CRALBP carries as endogenous ligands retinoids that are only known to function in vision, namely 11-cis-retinol and 11-cis-retinaldehyde and has only been found in tissues that respond to light (retina and pineal). Appropriate attention will be given to cloning and sequence analysis of the human CRALBP gene and to identification of regulatory regions responsible for tissue-specific expression. A concerted effort will also be made to identify the domains responsible for retinoid binding and interaction with retinal pigment epithelium (RPE) retinol dehydrogenase and retinyl ester synthase. The cellular localization of the protein will be confirmed by in situ techniques, the chromosomal location of the human gene determined and clinical linkage studies pursued. The possible involvement of CRALBP in hereditary retinal diseases such as retinitis pigmentosa is of major interest. A long-range goal is to define through genetic/protein engineering studies the structural features of the protein that are responsible for its high stereoselectivity and low photosensitivity. Regulatory regions of the gene will be characterized by primary RPE and Muller cell culture transfection CAT assays. CRALBP mRNA levels will be measured in RPE and Muller cell culture following treatment with various steroid hormones, growth factors and retinoids. The whole protein will be chemically synthesized in a limited number of peptides (16-30 residues/peptide) by automatic solid phase methods and anti-CRALBP peptide antibodies raised in rabbits. Functional domains will be probed by screening for function-blocking antibodies and peptides and by limited proteolysis. The combined results from the proposed research will provide a precise molecular framework in which questions concerning the normal function of CRALBP and the visual disorders it may be associated with can be answered in specific terms.