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. Additionally, we are also investigating the post-transcriptional regulation of RPE65 expression in RPE. In the past year we have made the following progress: a) In ongoing bioinformatics studies (in collaboration with NCBI/NLM) of chordate genomes into the evolutionary origins of RPE65, we discovered a new subfamily of carotenoid cleavage oxygenases (CCOs) in metazoans, the BCO2-like (BCOL) clade. These were found to be present in cephalochordate (including lancelets), nematode, and molluscan taxa. Distinctively, BCOL proteins lack the conserved PDPC(K) motif found in all previously characterized metazoan BCO proteins (e.g., BCO1, BCO2 and RPE65 in vertebrates, and numerous paralogs in other taxa). Phylogenetic analysis of CCOs in all kingdoms of life confirmed that the BCOL enzymes are an independent clade of ancient origin. We cloned one of the predicted lancelet BCOL proteins and analyzed it for carotenoid cleavage activity in a bacterial carotenoid expression system. We found it had activity similar to lancelet BCO2 proteins, although with a preference for cis-isomers. Our docking predictions correlated well with the cis-favored activity. The extensive expansions of the new animal BCOL family in some species (e.g., lancelet) suggests that the carotenoid cleavage oxygenase superfamily has evolved in the extremely high turnover fashion: the numerous losses and duplications of this family are likely to reflect complex regulation processes during development and interactions with the environment. These findings also serve to provide a rationale for the evolution of the important BCO-related outlier RPE65 retinol isomerase, an enzyme that does not utilize carotenoids as substrate or perform carbon-carbon double-bond oxidative cleavage. A manuscript describing these results was published in this reporting period. b) We completed a project to investigate palmitoylation of RPE65 cysteine(s), a controversial aspect of RPE65 biochemistry. Association with the endoplasmic reticulum (ER) membrane is a critical requirement for the catalytic function of RPE65. Several studies have investigated the nature of the RPE65-membrane interaction; however, complete understanding of its mode of membrane binding is still lacking. Previous biochemical studies suggest the membrane interaction can be partly attributed to S-palmitoylation, but the existence of RPE65 palmitoylation remains a matter of debate. We re-examined RPE65 palmitoylation, and its functional consequence in the visual cycle. We clearly demonstrated that RPE65 is post-translationally modified by a palmitoyl moiety, but this is not universal (about 25% of RPE65). By extensive mutational studies we mapped the S-palmitoylation sites to residues Cys112 and Cys146. Inhibition of palmitoylation using 2-bromopalmitate and 2-fluoropalmitate completely abolish its membrane association. Furthermore, palmitoylation-deficient C112 mutants are significantly impeded in membrane association. Finally, we showed that RPE65 palmitoylation level is highly regulated by lecithin: retinol acyltransferase (LRAT) enzyme. In the presence of all-trans retinol, LRAT substrate, there is a significant decrease in the level of palmitoylation of RPE65. Thus, our findings suggest that RPE65 is indeed a dynamically-regulated palmitoylated protein and that palmitoylation is necessary for regulating its membrane binding, and to perform its normal visual cycle function. A manuscript describing these results was submitted during this reporting period and is under review. c) We completed a study on a presumptive dominant-acting RPE65 mutation knock-in mouse model that we made by CRISPR/Cas9 technology. Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430 A>G, p.D477G mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice showed relatively undisturbed visual function with minimal retinal structural changes. However, KI/KI mouse retinae were more sensitive to light exposure and exhibited signs of degenerative features when subjected to light stress. We found that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affected appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrated that a splicing defect is associated with c.1430G pathogenesis, and therefore provides guidance in the therapeutic strategy for human patients. A manuscript describing these results was submitted during this reporting period and is under review. d) We continued a project investigating the post-transcriptional regulation of RPE65 expression that occurs in a variety of cell culture systems including primary RPE cell cultures and cell lines such as ARPE-19. We documented this in our original description of the RPE65 cDNA (Hamel et al, JBC, 1993) when we found that RPE65 protein expression decreased to zero in RPE primary cultures by 12 days after explantation, while levels of RPE65 mRNA remained relatively stable, and we hypothesized that it involved a post-transcriptional mechanism. In subsequent experiments (Liu and Redmond, ABB, 1998), we found that the 3 UTR of RPE65 mRNA played a role in this regulation, and contained a putative translation inhibition element (TIE) in the proximal 150 nt of the 3 UTR. More recently, our efforts to link this putative TIE to possible miRNA-mediated regulation were inconclusive. Our current efforts are directed towards elucidating whether the regulation is due to association of RPE65 mRNA with RNA-binding proteins, protecting it but sequestering it from ribosomal translation. We are using a number of approaches to address this question: protein binding to synthetic RNA, RNA pulldown, and density gradient fractionation of cellular RNA. Candidate proteins have been identified and are undergoing characterization.
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