Retinyl Ester Binding Proteins and the Visual Cycle

Harvard University (Medical School), Boston MA

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Abstract

[unreadable] 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). [unreadable] [unreadable]