Well over a half-century has passed since Wald showed that the photochemical cis-trans isomerization of the chromophore of rhodppsin is the primary event in vision. While substantial progress has been made in understanding how rhodopsin is regenerated after photoisomerization, there are still large gaps in our understanding of this process as part of the overall visual cycle. This proposal is focused on closing these gaps by continuing our studies on the chemical biology of the visual cycle. The critical trans-cis isomerization reaction in the visual cycle is dependent on a three-component system. Lecithin retinol acyl transferase (LRAT) is responsible for the esterificiation of vitamin A into all-trans-retinyl esters (tREs), the substrates for isomerohydrolase (IMH). IMH processes tREs into 11-cis-retinol. The third known component is RPE65, which acts as a chaperone for the highly hydrophobic tREs, allowing them to be processed by IMH. This grant is specifically concerned with extending our studies on LRAT and IMH, the two enzymes absolutely essential for visual pigment regeneration. LRAT is a multifunctional enzyme, which is essential for rhodopsin regeneration, and is the founder member of a large class of novel enzymes of largely unknown function. Because LRAT is physiologically important and novel, mechanistic and structural studies on the enzyme are warranted. We propose to (1) determine its three dimensional structure, (2) to characterize the nature of its multifunctional substrate binding site, and (3) to develop specific antagonists of the enzyme. Specific antagonists will be used to probe the multifunctional physiological roles of the enzyme and to serve as prototypes for the design of drugs used to limit the visual cycle for the treatment of macular degeneration. Our understanding of the chemical biology of IMH is considerably less advanced than that achieved with LRAT. We propose to identify mammalian IMH largely through the use of novel and specific affinity labeling agents of the enzyme. Several candidate proteins have already been identified. We further propose to express IMH in insect cells and fully purify it in order to establish its mechanism of action, and to eventually solve its three-dimensional structure. We further plan to quantitatively establish its role in rhodopsin regeneration. Finally, we plan to synthesize novel antagonists of IMH to aid in understanding its physiological role(s). Specific IMH antagonists will also be used to limit the visual cycle in approaches to the treatment of macular degeneration.
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