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 is 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 regulation of RPE65 expression in RPE. In the past year we have made the following progress: a) We are studying how RPE65 binds to smooth RPE endoplasmic reticulum (sER). RPE65 is an indispensable player in the retinoid visual cycle between the vertebrate retina and RPE. Although membrane association is critical for function, the underlying mechanism remains unclear. I a previous reporting period we found that the amphipathic (AH)-forming aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. These studies allow us to propose a working model for RPE65-membrane binding via a mechanism of “disorder-to-order” transition, and to provide a novel role for Cys palmitoylation. We are investigating the role of “disorder-to-order” transition in disease mechanisms by assessing how different pathogenic mutations in aa107-125, cataloged in the Leiden (Open) Variation Database (LOVD), affect binding to sER. By affecting substrate access such variants can affect enzymatic activity in a manner separate from catalytic activity. Known pathogenic variants inserted into GFP- AH107-125 were found to affect ER localization and/or stability. To complement biochemical and biophysical studies we generated mouse knockin models (C112Y and R118S) to study how these pathogenic variants affect in vivo physiology. Phenotypic analyses show that the C112Y homozygous mutant mouse has significantly reduced visual function. However, it still has recordable ERGs and reduced production of 11-cis retinal. The R118S appears to be a functional null based on preliminary phenotypic analysis. Manuscripts describing these results are being prepared for submission in the next reporting period. b) We continued a study on palmitoylation of BCO2 and BCO1 to see how palmitoylation may play a role in the structure and function of the other CCDs related to RPE65 in man and other mammals. All mammalian CCDs (RPE65, BCO1 and BCO2, as well as most other metazoan CCDs) 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 also loses palmitoylation in the presence of its substrates. We are continuing this work by studying the functional role of relocalization of BCO2 to the nucleus and its potential role in transcriptional regulation. To develop strategies for crystallization of BCO1, BCO2 and RPE65, in collaboration with a lab at NIAID, we expressed a secreted CCD protein, crystallized it and acquired a structure to a resolution of ~2.5 Å. We are currently attempting to get a structure containing a possible substrate or product (apocarotenal). These studies are in progress. c) We began a collaborative study to determine the molecular mechanism underlying the newly discovered c.1555G>A/ p.E519K mutation in RPE65, a presumptive dominant-acting RPE65 mutation that results in late-onset blindness. Recently identified in the Flanders region of Belgium in relatively large numbers it has been found in other parts of the world, including North America. While RPE65 mutations have been invariably recessively inherited, this variant, along with the previously identified c.1430A>G/p.D477G variant, has been reported to cause autosomal dominant retinitis pigmentosa (adRP) with a distinct late-onset pattern dystrophy. We determined that the enzymatic activity of the c.1555G>A/ p.E519K protein was reduced (but not abolished) compared to wildtype. In addition, we have generated a c.1555G>A/ p.E519K knockin mouse model to explore the in vivo phenotype. We are co-authors on a manuscript describing the c.1555G>A/ p.E519K mutation that was submitted and accepted for publication during this reporting period. We are continuing research on the RPE65 c.1430A>G mutation that is dominant in human by examining the effect of this mutation on splicing in RPE derived from patient induced pluripotent stem cell (iPSCs). Also, we are analyzing RPE derived from isogenic iPSC lines. We had earlier shown that “confused splicing” underlay the reduced expression of RPE65 mRNA in the mouse model, as well as in human in vitro cells. Our hypothesis has been that in humans because RPE65 transcription is so high the mutation causes “crashing” of the spliceosome. Because RPE65 transcription is much lower in mice, the c.1430A>G mutation is only evident in the homozygous knock-in. We have generated a mouse mutant of one of the spliceosomal machinery genes to cross with the c.1430A>G/p.D477G mouse to see if we can get a phenotype in the RPE65 c.1430A>G heterozygous mouse. These studies are in progress. d) We continued 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. The visual cycle is an important pathway in the retinal pigment epithelium (RPE) which regenerates 11-cis retinal chromophore for the retinal photoreceptors. Expression of RPE65 mRNA and protein levels are significantly lower in RPE cell culture models when compared to native RPE. This limits the use of these models to study the visual cycle. To determine the main drivers of RPE65 regulation we compared the transcriptional profiles of native and cell culture models of RPE with various levels of RPE65 expression. We also compared the levels of RPE65 expression between ARPE-19 cells grown in media supplemented with 1 mM pyruvate (PYR) or 10 mM nicotinamide (NAM). In addition, we performed experiments directed at transcriptional and translational regulation of RPE65. We found that RPE65 mRNA and protein expression is significantly higher in NAM media grown cells than PYR cells. Transfection of cells with a variety of different vectors containing RPE65 ORFs with different promoters, codon optimization, IRES, 3' UTRs, suggest that translational effects are less important than transcriptional status. Importantly, we found that feeding with rod outer segments (ROS) decreases RPE65 expression in NAM grown cells, suggesting that certain primary functions of the RPE (here, visual cycle and phagocytosis) are not positively linked. Analysis of differentially regulated microRNAs (miRs) provides a basis for this downregulation. It appears that the regulation of RPE65 expression in ARPE-19 cells, in particular, is multifactorial, involving primarily metabolic and transcriptional status of the cells, with translation of RPE65 mRNA playing a smaller role. A manuscript describing these results was submitted and was published in this reporting period.