Retinyl Ester Binding Proteins and the Visual Cycle

描述(申请人提供)：在视网膜色素上皮(RPE)中，通过将全反式视黄醇(维生素A)转化为11-顺式视黄醇(Al)所涉及的生化反应，完成视觉周期。其中一些关键步骤包括卵磷脂视黄醇酰基转移酶(LRAT)催化的维生素A的酯化反应，以卵磷脂为酰基供体生成疏水性全反式视黄酸酯，然后通过异麦芽水解酶将这些酯加工成11-顺式视黄醇。有关视觉周期运作的重要问题包括：确定参与视觉周期的维甲酸结合蛋白(RBPs)的完整库存，了解其如何调节，以及了解高度疏水的长链脂肪酸视黄酸酯是如何动员和加工的。RPE65已被证明是结合和动员疏水的全反式视黄酸酯(TRES)以供异麦芽水解酶(IMH)加工所必需的。已知RPE65的突变会导致一种形式的视黄醇变性。只有膜结合形式(MRPE65)才能立体特异性地结合Tres，而这种蛋白的可溶性形式(SRPE65)被证明能以高亲和力立体特异性结合维生素A。这两种形式的RPE65被LRAT相互转化，在这里起棕榈酰基转移酶的作用，并将棕榈酰基从mRPE65转移到维生素A或11-顺式视黄醇。我们建议RPE65外轮作为视觉周期运行中的一个重要的调节开关。控制元件揭示了棕榈酰化蛋白的新角色和功能，它根据mRPE65/sRPE65的相对水平在视觉周期中引导视黄醇流动。这一模型将使用生化和功能方法进行严格的测试。例如，将描述m和sRPE65的翻译后修饰的性质，并阐明LRAT介导的相互转换的分子酶学。我们将探讨mRPE65和sRPE65识别维甲酸的化学生物学基础。将确定sRPE65和mRPE65在视觉循环功能中的作用。除了已知的11-顺式维甲酸对IMH的反馈抑制外，RPE65周期是视觉周期操作中唯一已知的另一个控制元件。最后，mRPE65与TRES的立体特异性结合表明将会有同源的11-顺式-RE结合蛋白。特异性亲和生物素化方法成功地鉴定了mRPE65是一种tre结合蛋白，将用于表征11-顺式-RE结合同源物和11-顺式视黄酸酯水解酶(S)。

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).