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, and 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, known as Leber Congenital Amaurosis 2 (LCA2). LCA2 has been successfully treated by somatic RPE65 gene therapy. Disruption of the RPE-based vitamin A visual cycle blocking regeneration of visual pigment chromophore is the common phenotype shared by humans with RPE65 gene defects (LCA2) and the Rpe65 knockout mouse (overaccumulation of all-trans-retinyl esters and total absence of 11-cis retinal, resulting in extreme insensitivity to light). We have established a catalytic role for RPE65 in the synthesis of 11-cis retinol, identifying it as the long-sought visual cycle isomerohydrolase. We have also been studying beta-carotene 15,15'-monooxygenase (BCMO1), which we first identified based on similarity to RPE65. BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid cleaving enzymes. Because they share structural features, including identical residues in the catalytic assemblage, BCMO1 is a useful model for our mechanistic studies addressing RPE65. In the past year we have made the following progress: a) We identified inhibitors of RPE65 that could have future therapeutic benefit. We previously showed that RPE65 does not specifically produce 11-cis retinol only but also 13-cis retinol, supporting a carbocation or radical cation mechanism of isomerization. The intrinsic properties of conjugated polyene chains result in facile formation of radical cations in oxidative conditions. We hypothesized that such radical intermediates, if involved in the mechanism of RPE65, could be stabilized by spin traps. We tested a variety of hydrophilic and lipophilic spin traps for their ability to inhibit RPE65 isomerohydrolase activity. We found that the aromatic lipophilic spin traps such as N-tert-butyl-alpha-phenylnitrone (PBN), 2,2-dimethyl-4-phenyl-2H-imidazole-1-oxide (DMPIO) and nitrosobenzene (NB) strongly inhibit RPE65 isomerohydrolase activity in vitro, while non-aromatic or hydrophilic spin traps had no inhibitory activity. This supports the hypothesis of a radical intermediate in the enzymatic mechanism of RPE65. We also determined that the mode of inhibition was uncompetitive. As PBN is relatively non-toxic and well-tolerated and has been tested in clinical trials, it may provide a means to reduce RPE65 activity in vivo, without complete inhibition of activity, such as in Stargardt macular dystrophy and age-related macular degeneration where bisretinoid accumulation is a concern. A paper describing these results was published during this reporting period. We are also studying another class of RPE65 inhibitor, unrelated to spin traps. b) We extended our understanding of how the structure of RPE65 directs isomerization. We built a structural model of the substrate-bound form of RPE65 and used site-directed mutagenesis to analyze invariant aromatic residues for isomerase activity. Substrate docking predictions of wildtype and mutant RPE65 were modeled. Homology models reveal a large, hydrophobic cleft lined by aromatic residues. The cleft opens out to the surface, allowing a direct conduit for membrane-bound substrate to access the RPE65 catalytic core. We analyzed several cleft residues, mainly phenylalanines. Previous, unexpected, results from F103 mutants showed that RPE65 is a leaky isomerase that can produce not only 11-cis retinol, but also biologically irrelevant 13-cis retinol. This implicated a delocalized bond-order retinyl intermediate (carbocation or radical cation) in the RPE65 mechanism and has already guided the discovery of specific inhibitorrs of RPE65 (see above). We identified other residues, mutations of which also yielded enzymes preferentially producing 13-cis isomer. Substrate docking models concurred with these experimental results. We conclude that these aromatic residues constrain cleft shape and volume to favor production of 11-cis retinol. A temporal model for progression of isomerization by RPE65 can be built based on these findings. Thus, by strategic placement of aromatics to modulate the geometry and chemistry of the catalytic cleft, RPE65 has evolved from a carotenoid oxygenase to an isomerase role to drive the retinal visual cycle. c) We began a study to establish (or disprove, as the case may be) palmitoylation of RPE65 cysteine(s), a controversial aspect of RPE65 biochemistry. Different groups have used mass spectrometry to definitively establish that RPE65 is palmitoylated, or that it is not. Clearly, only one of these alternatives is true. We are using a bioorthogonal method to determine if RPE65 is acylated by metabolic labeling in a physiologically relevant cell culture model. Existence of labeled cysteine(s) will be established by mass spectrometry of RPE65 peptides. We are also beginning experiments to discern how the retinoid visual cycle integrates with overall lipid metabolism in the RPE. d) A project to study the role of A2E in RPE cytotoxicity for Stargardt disease and to study possible therapies was initiated in collaboration with Dr. Brian Brooks and Dr. Nataly Strunnikova (OGVFB). While the toxic effects of A2E accumulation have been shown for high A2E concentrations in a mouse model of recessive Stargardt's macular degeneration, the literature on A2E toxicity to RPE cells at low micromolar concentration varies from DNA protective effects to mitochondrial damage and apoptosis. We believe that this variability may be partially explained by differences in how A2E is synthesized and delivered in these experiments. We found a significant lysosomal pH change after treatment with low micromolar concentrations of A2E fluoroacetate using a pH sensitive lysosensor. However, this treatment with A2E was not apoptotic or proapoptotic. We are investigating further how the use of different counterions affects lysosomal alkalinization and other effects in RPE cells. Targeting this pathway may prevent the deleterious effects of lysosomal alkalinization. Also, we developed a mass spectrometry method to quantitate A2E and applied this to the quantitation of A2E from mouse and human tissues (in collaboration with Crouch/Ablonczy lab at MUSC, Charleston, SC). e) To complement prior work on pathogenic human RPE65 hypomorphic mutations, such as P25L, we have generated, in collaboration with the NEI Genetic Engineering Core, a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. It is anticipated that these will provide important insight into the variability of RPE65-deficient phenotypes, in comparison with the extreme case of the knockout. In particular, we hope to provide insight into the slower progression of the retinal degeneration such as seen in less severe cases of human RPE65 mutations. They also will provide animal models to test pharmacologic strategies. We have established three knock-in lines. To abrogate interference with transcription and/or mRNA processing due to the neo selection cassette, resulting in an effectively null phenotype, we had to breed all three knock-ins with the Zp3-Cre line to remove the neo cassette. The three lines are now being phenotyped. Preliminary data, obtained in collaboration with the NEI Visual Function Core, reveals pronounced changes in the kinetics of regeneration in these knockins, consistent with our predictions.
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