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 cleavage dioxygenases (CCDs), 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 how RPE65 binds to smooth RPE endoplasmic reticulum (sER). RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, while palmitoylation at C112 also plays a role. We identified the mechanism of membrane recognition and binding by RPE65. We found that binding of an aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic a-helical (AH) structure but that this transition was automatic in the C112-palmitoylated peptide. We found that this AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, we found that aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly showed AH-membrane insertion, supporting our experimental findings. Collectively, these studies allowed us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation. A manuscript describing these results was submitted during the last reporting period and was published during the current reporting period. We are continuing this work by assessing how different pathogenic mutations in aa107-125 affect binding to sER. 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 prior reporting periods we found that BCO2 was also palmitoylated but, in contrast, BCO1 was found not to be palmitoylated. BCO2 lost palmitoylation in the presence of its substrates beta-carotene and different xanthophylls. 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). Our finding that BCO2 loses palmitoylation in presence of xanthophylls and its translocation into the nucleus suggests a possible mechanism for transport of lutein/lutein cleavage products to the nucleus to affect transcriptional regulation. We are continuing this work by studying the functional role of relocalization of BCO2 to the nucleus and its potential role in transcriptional regulation. We are also dissecting the structural/functional role of BCO2 palmitoylation on itssubcellular localization. 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. 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 several 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.