Retinyl Ester Binding Proteins and the Visual Cycle

描述（由申请人提供）：视觉循环在视网膜色素上皮（RPE）中通过参与将全反式视黄醇（维生素A）加工成11-顺式视黄醇（al）的生化反应完成。其中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酯化反应，以卵磷脂为酰基供体生成疏水全反式视黄醇酯，然后通过异构水解酶将这些酯加工成11-顺式视黄醇。关于视觉循环操作的重要问题包括识别参与循环的类视黄醇结合蛋白（rbp）的完整清单，了解它是如何调节的，以及了解高度疏水的长链脂肪酸视黄醇酯是如何动员和加工的。RPE65已被证明是结合和动员疏水全反式视黄酰基酯（tREs）的必要条件，以供异构体水解酶（IMH）加工。已知RPE65的突变会导致视黄醇变性的一种形式。只有膜相关形式（mRPE65）与tREs立体特异性结合，而该蛋白的可溶性形式（sRPE65）被证明具有高亲和力的立体特异性结合维生素A。这两种形式的RPE65通过LRAT相互转化，在这里作为棕榈酰转移酶，并将棕榈酰基团从mRPE65转移到维生素a或11-顺式视黄醇。我们提出RPE65本轮是视觉周期运行中必不可少的调控开关。控制元件揭示了棕榈酰化蛋白的新作用和功能，通过mRPE65/sRPE65的相对水平来指导视觉周期中的类视黄醇流。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。探讨mRPE65和sRPE65识别类维生素a的化学生物学基础。sRPE65和mRPE65在视觉循环功能中的作用将被确定。除了已知的11-顺式类维甲酸对IMH的反馈抑制外，所提出的RPE65周期代表了视觉周期操作中唯一已知的其他控制因素。最后，mRPE65对tREs的立体特异性结合表明，将存在同源11-顺式re结合蛋白。特异性亲和生物素化方法已被证明成功地鉴定了mRPE65作为tRE结合蛋白，将被用于表征11-顺式re结合同源物和11-顺式视黄酰基酯水解酶。

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).