Retinyl Ester Binding Proteins and the Visual Cycle

视黄酯结合蛋白和视觉周期

• 批准号: 6855567
• 负责人: ROBERT R RANDO
• 金额: $ 42.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855567
• 关键词: Rana; biochemistry; cysteine; fatty acylation; laboratory mouse; mass spectrometry; membrane proteins; palmitates; posttranslational modifications; protein structure function; retinoid binding proteins; retinoids; stereochemistry; vision

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）：通过将全反式视黄醇（维生素A）加工成11-顺式视黄醇（al）的生化反应，在视网膜色素上皮（RPE）中完成视觉周期。一些关键步骤包括卵磷脂视黄醇酰基转移酶（LRAT）介导的使用卵磷脂作为酰基供体的维生素A的酯化以生成疏水性全反式视黄酯，随后通过异构水解酶加工这些酯以形成11-顺式-视黄醇。关于视觉周期的操作的重要问题包括识别参与周期的类维生素A结合蛋白（RBP）的完整库存，了解它是如何调节的，以及了解高度疏水性的长链脂肪酸视黄酯是如何被动员和处理的。RPE 65已被证明是必不可少的结合和动员的疏水性全反式视黄酯（tRE）的加工异构水解酶（IMH）。已知RPE 65中的突变会导致一种形式的视黄基变性。只有膜结合形式（mRPE 65）立体特异性结合tRE，并且该蛋白质的可溶形式（sRPE 65）显示出以高亲和力立体特异性结合维生素A。两种形式的RPE 65通过LRAT相互转化，在此作为棕榈酰转移酶，并将棕榈酰基团从mRPE 65转移至维生素A或11-顺式-视黄醇。我们提出了一个RPE 65 epicardium作为一个必不可少的监管开关的视觉周期的运作。控制元件揭示了棕榈酰化蛋白质的新作用，并通过根据mRPE 65/sRPE 65的相对水平指导视色素在视觉周期中的流动来发挥功能。该模型将使用生物化学和功能方法进行严格测试。例如，将描述m和sRPE 65的翻译后修饰的性质，并阐明LRAT介导的相互转化的分子酶学。将探索mRPE 65和sRPE 65识别类维生素A的化学生物学基础。将确定sRPE 65和mRPE 65在视觉周期功能中的作用。除了已知的11-顺式维甲酸对IMH的反馈抑制外，所提出的RPE 65循环代表了视觉循环操作中唯一的其他已知控制元件。最后，mRPE 65对tRE的立体特异性结合的证明表明将存在同源11-顺式RE结合蛋白。将采用特异性亲和生物素化方法（已证明成功鉴定mRPE 65为tRE结合蛋白）来表征11-顺式-RE结合同源物和11-顺式-视黄酯水解酶。