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 the target of successful somatic RPE65 gene therapy trials. Disruption of the RPE-based vitamin A visual cycle for isomerization of vitamin A to regenerate 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. We postulate that BCMO1 and RPE65 share a similar underlying mechanism of action. Because they share structural features, including identical residues in the catalytic assemblage, we have found BCMO1 to be 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. RPE65 is the visual cycle retinol isomerase, converting all-trans retinyl esters into 11-cis retinol in the critical step of chromophore regeneration. However, the precise enzymatic mechanism underlying this conversion has been a matter of debate for many years. Two alternative mechanisms, with different predicted specific outcomes, have been proposed. One mechanism predicts a specific isomerization to 11-cis retinol, while the other allows for the production of isomers other than 11-cis. We reasoned that the mechanism of RPE65 would be consistent with an overall mechanism for the carotenoid oxygenase family, of which it is a member. The availability of crystal structures for carotenoid oxygenases, including RPE65, allowed us to investigate the roles of aromatic residues in the predicted substrate-binding site in the mechanism. Using this approach, we found that RPE65 is not an inherently 11-cis specific isomerase and can produce 13-cis retinol as robustly as 11-cis retinol. Moreover, single-residue mutations can modulate RPE65 activity toward a 13-cis specific outcome. Taken together, these results are consistent with a carbocation or a radical cation intermediate-mediated mechanism of isomerization, either of which can explain synthesis of both 11- and 13-cis retinols. It also allows rational exploration of possible inhibitors of RPE65. In the past year, to determine if radical cation intermediates are involved in this mechanism, we tested whether spin-trap compounds could quench RPE65 isomerase activity. In vitro visual cycle assays in 293-F cells were incubated with 0-400 micromolar concentrations of several spin-trap compounds. The spin-trap alpha-phenyl N-tert-butylnitrone (PBN), but not other spin-traps, strongly inhibited RPE65 activity but did not affect RPE65 expression, suggesting a direct quenching effect on isomerase activity. Thus, we concluded that radical cation intermediates are involved in the mechanism of RPE65. We also determined that the mode of inhibition was uncompetitive. This means that while the degree of inhibition by an uncompetitive inhibitor increases with substrate concentration, it never completely blocks turnover at low substrate concentrations. This is important in the context of a potential use of PBN as a modulator of RPE65 and the visual cycle since it would allow for lowered but not abolished formation of 11-cis retinoids (which would cause blindness). 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. We are also studying another class of RPE65 inhibitor. b) BCMO1, RPE65 and related enzymes share histidine and acidic residues that are involved in iron coordination crucial to activity of these enzymes, but other crucial aspects of the mechanism (substrate binding, intermediate formation, inhibitor relationships, etc.) remain unclear. In the past year, we conducted experiments that identified a novel inhibitor of BCMO1. Fenretinide (4-hydroxy(phenyl)retinamide;4-HPR), a synthetic retinoid derivative, shows anticancer potential in clinical trials for chemoprevention and treatment of breast cancer. Common side effects associated with fenretinide treatment include night-blindness, which is reversible upon cessation of treatment, initially attributed to inhibition of RBP4 (retinol binding protein 4) binding to retinol and consequent impaired retinol transport. Fenretinide is also being tested in clinical trials as a therapy for Stargardt maculopathy and for age-related macular degeneration (AMD). However, fenretinide affects Rbp4-/- animals similarly to wild type mice, and, thus, the reason for the effects of fenretinide remains elusive. It has been suggested that beta-carotene monooxygenase 1 (BCMO1) might supply all-trans retinal as an accessory source of vitamin A for the visual cycle. We found that fenretinide is a strong inhibitor of mouse BCMO1 (IC50= 1.2 microM), acting non-competitively. In contrast, other retinoids, such as retinyl palmitate and retinyl acetate, as well as other biologically active aromatic compounds (capsaicin and resveratrol, and the amino analog of fenretinide) do not substantially inhibit BCMO1 activity. To define the mechanism of inhibition more clearly, we deleted portion of an inter-strand loop of BCMO1, that we term the metazoan loop, to generate BCMO1del336-345. This mutant had impaired enzymatic activity, but was not substantially inhibited by fenretinide. Using computer docking we concluded that binding of fenretinide to BCMO1 is determined by two hydrogen bonds. Thus, we found that fenretinide is a strong non-competitive inhibitor of BCMO1 that does not bind in the active site, but at the metazoan loop. We also show that the integrity of the metazoan loop influenced binding of this retinoid. Our data point to a mechanism of fenretinide-induced night blindness through inhibition of BCMO1 as a local supplier of vitamin A to the visual cycle. c) To complement prior work on hypomorphic mutations, such as P25L, in human disease we generated 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 the knock-in line for three constructs. Preliminary phenotyping of one of the lines, H182Y, revealed likely interference with transcription and/or mRNA processing due to the neo selection cassette, resulting in an effectively null phenotype. This line is being bred with the Zp3-Cre line to remove the neo cassette to relieve this interference. The remaining 2 lines are entering phenotyping. They will also be crossed to Zp3-Cre to remove the neo cassette.
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