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Retinyl Ester Binding Proteins and the Visual Cycle

视黄酯结合蛋白和视觉周期

基本信息

批准号：
7176097
负责人：
ROBERT R RANDO
金额：
$ 35.89万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-01-01 至 2007-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7176097
关键词：
11 cis Retinal 11-cis-Retinol Affinity Affinity Labels All-Trans-Retinol Anabolism Back Binding Binding Proteins Biochemical Biochemical Reaction Biochemistry Biological Biotin Biotinylation Chemicals Cysteine Elements Enzymatic Biochemistry Enzymes Equipment and supply inventories Esterification Esters Future Goals Grant Indium Lecithin Light Adaptations Mediating Membrane Methods Modeling Modification Molecular Mutation Nature Operative Surgical Procedures Pathway interactions Post-Translational Protein Processing Process Proteins RPE65 protein Regulation Relative (related person)Research Research Personnel Retinoids Retinol Binding Proteins Role Specificity Structure of retinal pigment epithelium Testing Transferase Vitamin A Work affinity labeling base chromophore ebelactone A feeding long chain fatty acid novel palmitoylation programs protein function retinyl esterase stoichiometry visual cycle

项目摘要

DESCRIPTION (provided by applicant): The visual cycle is completed in the retinal pigment epithelium (RPE) by those biochemical reactions involved in the processing of all-trans-retinol (vitamin A) into 11-cis-retinol(al). Some of the critical steps include the lecithin retinol acyl transferase (LRAT) mediated esterification of vitamin A using lecithin as the acyl donor to generate hydrophobic all-trans-retinyl esters followed by the processing of these esters to form 11-cis-retinol by isomerohydrolase. Significant questions concerning the operation of the visual cycle include the identification of the full inventory of retinoid binding proteins (RBPs) involved in the cycle, an understanding of how it is regulated, and an understanding of how the highly hydrophobic long-chain fatty acid retinyl esters are mobilized and processed. RPE65 has been shown to be essential for the binding and mobilization of the hydrophobic all-trans-retinyl esters (tREs) for processing by isomerohydrolase (IMH). Mutations in RPE65 are known to cause a form of retinyl degeneration. It is only the membrane associated form (mRPE65) which stereospecifically binds tREs and the soluble form of this protein (sRPE65) is shown to stereospecifically bind vitamin A with high affinity. The two forms of RPE65 are interconverted by LRAT, acting here as a palmitoyl transferase, and transferring a palmitoyl group from mRPE65 to vitamin A or 11-cis-retinol. We propose an RPE65 epicycle as an essential regulatory switch in the operation of the visual cycle. The control element reveals new roles for palmitoylated proteins and functions here by directing retinoid flow in the visual cycle depending on the relative levels of mRPE65/sRPE65. This model will be rigorously tested using biochemical and functional approaches. For example, the nature of the post-translational modifications of m and sRPE65 will be described and the molecular enzymology of the LRAT mediated interconversion will be elucidated. The chemical biological basis of mRPE65 and sRPE65 recognition of retinoids will be explored. The roles of sRPE65 and mRPE65 in visual cycle function will be determined. Aside from the known feed-back inhibition of IMH by 11-cis-retinoids, the proposed RPE65 cycle represents the only other known control element in the operation of the visual cycle. Finally, the demonstration of the stereospecific binding of tREs by mRPE65 suggests that there will be cognate11-cis-RE binding proteins. Specific affinity biotinylation methods, which proved to be successful in the identification of mRPE65 as a tRE binding protein, will be adapted to characterize the 11-cis-RE binding cognates and 11-cis-retinyl ester hydrolase(s).

描述（由申请人提供）：视觉循环是在视网膜色素上皮（RPE）中通过将全反式视黄醇（维生素 A）加工成 11-顺式视黄醇（a1）所涉及的生化反应而完成的。一些关键步骤包括卵磷脂视黄醇酰基转移酶 (LRAT) 介导的维生素 A 酯化，使用卵磷脂作为酰基供体，生成疏水性全反式视黄酯，然后通过异构水解酶处理这些酯以形成 11-顺式视黄醇。有关视觉循环运作的重要问题包括识别循环中涉及的类视黄醇结合蛋白 (RBP) 的完整库存、了解其调节方式以及了解高度疏水性长链脂肪酸视黄酯如何动员和加工。 RPE65 已被证明对于疏水性全反式视黄酯 (tRE) 的结合和动员以供异构水解酶 (IMH) 加工至关重要。已知 RPE65 突变会导致某种形式的视黄质变性。只有膜相关形式 (mRPE65) 能够立体特异性地结合 tRE，而该蛋白的可溶形式 (sRPE65) 则能够以高亲和力立体特异性地结合维生素 A。 RPE65 的两种形式通过 LRAT 相互转化，在此充当棕榈酰转移酶，并将棕榈酰基从 mRPE65 转移为维生素 A 或 11-顺式-视黄醇。我们提出 RPE65 本轮作为视觉循环运行中的重要调节开关。该控制元件通过根据 mRPE65/sRPE65 的相对水平引导视觉周期中的类视黄醇流动，揭示了棕榈酰化蛋白的新作用和功能。该模型将使用生化和功能方法进行严格测试。例如，将描述 m 和 sRPE65 翻译后修饰的性质，并阐明 LRAT 介导的相互转化的分子酶学。将探讨 mRPE65 和 sRPE65 识别类视黄醇的化学生物学基础。将确定 sRPE65 和 mRPE65 在视觉周期功能中的作用。除了已知的 11-顺式-维甲酸对 IMH 的反馈抑制之外，所提出的 RPE65 循环代表了视觉循环操作中唯一已知的其他控制元件。最后，mRPE65 对 tRE 的立体特异性结合的证明表明将会存在同源 11-cis-RE 结合蛋白。特异性亲和生物素化方法已被证明可以成功地将 mRPE65 鉴定为 tRE 结合蛋白，该方法将适用于表征 11-cis-RE 结合同源物和 11-cis-视黄酯水解酶。