Molecular Biology Of Outer Retina-specific Proteins

National Eye Institute

Investigators

Linked publications & trials

Abstract

We are studying RPE-specific mechanisms, at both the regulatory and functional levels, and have been studying the structure, 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, how its structure contributes to this mechanism, as well as its regulation and activity in the context of photoreceptor development and in disease. We are also studying structural and functional aspects of 2 related carotenoid oxygenases, BCO1 and BCO2, crucial for other aspects of carotenoid/retinoid metabolism, both ocular and systemic. The role of RPE65 in inherited retinal dystrophies in being studied using genetically modified mice as well as with RPE cells derived from induced pluripotent stem cells (iPSCs) from affected patients. 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) We are continuing a project investigating the functional role of 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. Our published findings to date show 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. Several studies have investigated the nature of the RPE65-membrane interaction; however, complete understanding of its mode of membrane binding is still lacking. We are continuing this work by studying the biochemical and biophysical role of palmitoylation in RPE65, its structural context in ER membrane binding, and the important role of the -PDPCK- motif and associated sequences in membrane association. An important aspect of this work is being completed, and a manuscript describing the results is being readied for submission during the upcoming reporting period. b) We continued to investigate palmitoylation of BCO2 to study the possibility that palmitoylation may play a role in the structure and function of the other related carotenoid oxygenases in man and other mammals. All mammalian carotenoid oxygenases (RPE65, BCO1 and BCO2, as well most other metazoan carotenoid oxygenases, contain the -PDPCK- motif, the cysteine of which is post-translationally modified in RPE65. In the prior reporting period we found that BCO2 was also palmitoylated but lost this palmitoylation in the presence of its substrate beta-carotene. In contrast, BCO1 was found not to be palmitoylated. We investigated the potential role xanthophylls play in BCO2 palmitoylation. An extensive body of work has documented the antioxidant role of xanthophylls (lutein and zeaxanthin) in human health and specifically how they provide photoprotection in human vision. More recently, evidence is emerging for the transcriptional regulation of antioxidant response by lutein/lutein cleavage products, similar to the role of beta-carotene cleavage products in the modulation of retinoic acid receptors. Supplementation with xanthophylls also provides additional benefits for the prevention of age-related macular degeneration (AMD). Mammalian BCO2 asymmetrically cleaves xanthophylls as well as beta-carotene in vitro. We had demonstrated in the previous reporting period that mouse BCO2 (mBCO2) is a functionally palmitoylated enzyme and that it loses palmitoylation when cells are treated with -carotene. We used the same acyl-RAC methodology and confocal microscopy to elucidate palmitoylation and localization status of mBCO2 in the presence of xanthophylls. We created large unilamellar vesicle-based nanocarriers for the successful delivery of xanthophylls into cells. We found that, upon treatment with low micromolar concentration of lutein (0.15 M), mBCO2 is depalmitoylated and showed partial nuclear localization (38.00 0.04%), while treatment with zeaxanthin (0.45 M) and violaxanthin (0.6 M) induced depalmitoylation and protein translocation from mitochondria to a lesser degree (20.00 0.01% and 35.00 0.02%, respectively). Such a difference in the behavior of mBCO2 toward various xanthophylls and its translocation into the nucleus in the presence of various xanthophylls suggests a possible mechanism for transport of lutein/lutein cleavage products to the nucleus to affect transcriptional regulation. Manuscripts describing these results were published during this reporting period. We are continuing this work by studying the functional role of relocalization of BCO2 to the nucleus and its potential role in transcriptional regulation. c) We continued a study to determine the molecular mechanism underlying the c.1430A>G/ p.D477G mutation in RPE65, a presumptive dominant-acting RPE65 mutation that results in blindness. While RPE65 mutations have been invariably recessively inherited, this particular mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP) with features resembling choroideremia. In the previous reporting period, we completed a study on a c.1430A>G/ p.D477G knock-in mouse model that we made by CRISPR/Cas9 technology. Our data demonstrated that a splicing defect is associated with c.1430G>A pathogenesis, and therefore provide insights in the therapeutic strategy for human patients and a manuscript describing these results was published in the last reporting period. We are following up this study by examining the effect of this mutation on splicing in RPE derived from induced pluripotent stem cell (iPSCs) from patients with the RPE65 c.1430A>G point mutation. Also, we are analyzing RPE derived from introduction of the point mutation via CRISPR/Cas9 in isogenic iPSCs. Comparison of these two lines of RPE cells (patient derived cells and isogenic point-mutated cells) will allow us to perform a differential analysis on the effect of the point mutation. In addition to these studies to establish mechanism, we have begun a collaboration with NCATS to develop a potential small-molecule therapy as the FDA-approved gene-replacement therapy is not indicated for dominant RPE65 dystrophy. These studies are in progress. d) We continued a project investigating the reduced transcriptional/translational expression of RPE65 that occurs in a variety of cell culture systems including primary RPE cell cultures and cell lines such as ARPE-19. We and others have found that RPE65 protein expression is completely absent in cultured RPE cell lines (e.g., ARPE-19, etc.) and most other RPE cultured cells. RPE65 transcription, however, occurs at a much-reduced level in these cultured cells compared to native RPE. The level of RPE65 transcription depends on the particular cell model and on culture conditions being analyzed. 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. A manuscript describing these results is being readied for submission during the upcoming reporting period. e) We continued a study to express/purify RPE65, BCO1 and BCO2 for structural analysis. Historically these proteins have been difficult to express and purify. The structural analyses include techniques such as small-angle X-ray scattering (SAXS) analysis, cryo-electron microscopy (cryoEM), nuclear magnetic resonance (NMR) analysis, as well as crystallization. These projects are being done in collaboration with NIH and extramural labs. These studies are in progress.