We are studying RPE-specific mechanisms, at both the regulatory and functional levels, and have been studying the function and regulation of RPE65, the key retinol isomerase enzyme of the visual cycle. Current work is focused on establishing the molecular mechanism of RPE65 catalysis, as well as its regulation and activity in the context of photoreceptor development and in disease. We are also studying the effects of bisretinoid byproducts of the visual cycle (e.g., A2E) on RPE lysosomal metabolism. In the past year we have made the following progress: a) We have, over the past few reporting periods, focused on determining how RPE65 catalyzes all-trans to cis isomerization of retinol, supporting a retinyl cation-mediated mechanism. We are currently addressing a further aspect of the complex mechanism of RPE65, that of the O-alkyl bond cleavage that results in leaving of the fatty acid moiety. We hypothesize that this is the acquired primary activity of RPE65, with cis isomerization being a secondary but crucial ancillary activity. We are focusing on i) defining the mechanism of O-alkyl cleavage and ii) determining the fate of the palmitate product. In this reporting period we have tested possible mechanisms for cleavage and fate of the palmitate product to answer these questions. In this reporting period we completed a project that identified the lipid analog triacsin C as an inhibitor of RPE65. We discovered that triacsin C, an inhibitor of ACSLs, potently competitively inhibited RPE65 isomerase activity in cellulo (IC50=500 nM). We confirmed that triacsin C competes directly with all-trans-retinyl ester (atRE) by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 M atRE. As triacsin C lacks structural features comparable with retinoids it probably competes with binding of the acyl moiety of atRE. These results were published in this reporting period. b) We continued a project to 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. Two separate approaches are being used to validate the presence or absence of a palmitoyl group. We have established that palmitoylation of RPE65 occurs. However the level of palmitoylation does not suggest a structural or permanent acylation of the RPE65. Rather, it is either dynamic or adventitious. Current work is focused on dissecting the functional relevance of RPE65 palmitoylation. In addition, we continue to conduct site-directed mutagenesis experiments to dissect the role of various key residues of RPE65 in aspects of its catalytic mechanism. c) In experiments published in the last reporting period, we analyzed the phenotype of the P25L hypomorphic knock-in of the mouse Rpe65 gene. This models the mild phenotype of a homozygous P25L LCA2 patient with well-preserved cone vision. Preserving cone function is a key concern in managing RPE65 retinal dystrophy, and an important objective of RPE65 gene therapy. In this reporting period we continued to use the P25L knock-in mouse to further delimit the minimum level of RPE65 required for cones. By in vivo titration of chromophore turnover, we aim to further determine effects on retinal physiology and cone survival/function, and to determine a minimum level of chromophore turnover to maintain cone viability and function. Experiments were performed with mice having one P25L knockin allele (KI) on a knockout (KO) background, i.e., KI/KO mice. Under regular vivarium conditions, the KI/KO mice, just as the KI/KI mice, have a close to wildtype phenotype. This suggests that a level of less than 10% wildtype expression of RPE65 is still capable of maintaining proper cone viability, at least under regular vivarium light conditions (100 lux). Experiments are being designed to expose KI/KI and KI/KO mice to higher but moderate levels of light (1000 lux) to further stress the visual cycle. In addition, we are also studying D477G (a putative dominant-acting RPE65 mutation) knock-in mice that we have made by CRISPR/Cas9 technology. The human D477G phenotype has aspects similar to choroideremia, and is quite distinct from the usual recessive RPE65 phenotypes. However, the dominant acting nature of the human disease is not reproduced in the mouse model (i.e., no phenotype in KI/+ mice), instead a complex interaction is revealed in KI/KI animals. Similar to P25L mice, the D477G mice have close to wildtype phenotype in homozygous state. Further experiments (similar to light-stress experiments being planned for the P25L model) will be done to better understand the KI/KI phenotype. In addition, human RPE cell experiments are planned to help unravel the complex phenotype. d) We completed a project aimed at resolving the pleiotropic effects of fenretinide on several enzymes (beta-carotene oxygenase 1 (BCO1), stearoyl-CoA desaturase 1 (SCD1), and dihydroceramide 4-desaturase 1 (DES1) by analyzing the effects of two major physiological metabolites of fenretinide. Fenretinide (N-4-hydroxyphenylretinamide (4-HPR)) is a synthetic retinoid that forms a tight complex with plasma retinol-binding protein (RBP4) and thus interferes with RBP4-transthyretin (TTR) complex formation and retinol transport. Fenretinide also prevents insulin resistance in mice, unrelated to its effect on RBP4-TTR. As fenretinide has been proposed as a therapy for age-related macular degeneration (AMD), its pleiotropic effects are a cause for concern. The effect of two physiological metabolites of fenretinide, N-4-methoxyphenylretinamide (MPR), and 4-oxo-N-(4-hydroxyphenyl)retinamide (3-keto-HPR), and the non-retinoid RBP4 ligand A1120, on BCO1, SCD1 and DES1 activity was determined in ARPE-19 cells. The activities of the three enzymes are inhibited by the parent fenretinide. We found that 3-keto-HPR is more potent than fenretinide for all three enzymes and may mediate most of the in vivo beneficial effects of fenretinide. While MPR does not affect SCD1 and DES1 activity, it is a potent specific inhibitor of BCO1. In contrast, the non-retinoid RBP4-specific ligand A1120 did not affect any of the studied enzymes. We concluded that administration of 3-keto-HPR instead of fenretinide would be preferential if inhibition of SCD1 or DES1 activity is the goal, while MPR is a specific BCO1 modulator. On the other hand, as it does not inhibit any of the enzymes, we confirm that A1120 appears to only mediate effects on RBP4. A manuscript describing these results was submitted late in this reporting period and is currently under review.
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