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 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 closely related to that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. In the past year we have made the following progress: a) We have identified the regulatory region of the RPE65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the RPE65 gene. b) Two collaborative efforts showed a direct relationship between the level of RPE65 expression in mice and susceptibility to retinal light damage. Rpe65 knockout mice or those with a less active variant are resistant to light-induced retinal damage. c) Analysis of aging Rpe65 knockout mice (up to two years old) show a dramatically lowered accumulation of the age-related pigment lipofuscin (an irreversible by-product of vitamin A associated with age-related macular degeneration) compared to wildtype. d) We have identified from mouse the first mammalian beta-carotene 15,15'-dioxygenase (beta-CD), a crucial enzyme in development and metabolism that governs the de novo entry of vitamin A from plant-derived beta-carotene. This protein is a homologue of RPE65 and a member of a newly emerging diverse family of enzymes that includes the plant 9-cis-epoxycarotenoid cleavage enzyme and bacterial lignostilbene dioxgenases. e) Gene vector constructs are being made in Adeno-associated virus and will be tested for gene transfer rescue of the knockout mouse.
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| Lyubarsky, Arkady L; Savchenko, Andrey B; Morocco, Sarah B et al. (2005) Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye. Biochemistry 44:9880-8 |
| Redmond, T Michael; Poliakov, Eugenia; Yu, Shirley et al. (2005) Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci U S A 102:13658-63 |
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