Molecular Biology Of Outer Retina-specific Proteins

National Eye Institute

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Abstract

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. Disruption of the RPE-based vitamin A visual cycle metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (beta-CM). Beta-CM is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. In the past year we have made the following progress: a) The role of residues conserved in all members of the carotenoid-cleavage enzyme family (including beta-CM and RPE65) has been further investigated by site-directed mutagenesis of putative metal binding residues in beta-CM. The data demonstrate a crucial role in enzymatic activity for histidine and acidic residues hypothesized to be involved in metal coordination. Mutation of the 4 conserved histidine and a conserved glutamate residue results in loss of iron-binding as shown by inductively coupled plasma atomic emission spectrometry. Mutation of certain other non-absolutely conserved residues results in impaired but active enzyme, chiefly resulting in Vmax changes of mutant enzymes. b) RPE65 contains two potential caveolin-interaction domains that are conserved in all species and are functionally active. Caveolin-1 is a "scaffolding" protein known to be involved in membrane trafficking, lipid transport and in signal transduction. Its interaction with RPE65 may involve some or all of these roles. We have found that RPE65 binds to both the scaffolding domain and the C-terminal domain of caveolin-1. This interaction, along with the previously shown interaction with phospholipids, may allow for the association of RPE65 with membrane anchored visual cycle components and provides a new focus for study of the visual cycle complex. To investigate such possibilities, caveolin knockout mice have been obtained and are being tested for phenotypic changes in vision, etc. c) Transgenic mice bearing transgenes incorporating mutations in RPE65 identified as pathogenic in humans have been bred onto the Rpe65 knockout background and are being tested as to their phenotypes. Several different lines have been generated. In this way it is hoped to generate models for RPE65-related retinal dystrophy that are not null (like the knockout mouse and the Briard dog) d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. Expression clones for these factors are being tested for their effect on activation of the RPE65 promoter. e) Collaboration continues on the rescue of the Rpe65 knockout mouse phenotype and the Briard dog RPE65 dystrophy by AAV-mediated gene transfer.