Molecular Biology Of Outer Retina-specific Proteins

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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 splicing defect that is associated with pathogenesis of the RPE65 c.1430A>G, p.D477G mutation in mouse and human RPE. Additionally, we are also investigating the post-transcriptional regulation of RPE65 expression in RPE and have begun a project on the mechanisms of translational pausing in SARS-CoV-2 spike protein and its potential role in COVID-19. In the past year we have made the following progress: a) We have been investigating 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 findings, to date, 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 published during the last reporting period. We are continuing this work by studying the biochemical and biophysical role of palmitoylation in RPE65. This work is in progress. In parallel with our studies on palmitoylation in RPE65, we studied the possibility that palmitoylation may play a role in the structure and function of the other related carotenoid oxygenases (BCO1 and BCO2) present in man and other mammals. All mammalian carotenoid oxygenases (CCOs): RPE65, BCO1 and BCO2, as well as the vast majority of other metazoan carotenoid oxygenases, contain the -PDPCK- motif, the cysteine of which is post-translationally modified in RPE65 and is likely to be associated with palmitoylation of other animal CCOs. We found that, in addition to RPE65, beta-carotene oxygenase 2 (BCO2) is also a palmitoylated protein. As with RPE65, we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate beta-carotene. We found that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate beta-carotene. In addition, we found that metazoan CCOs lacking the motif (e.g., Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora apocarotenoid oxygenase-like protein (ACOL)) are not palmitoylated. Parenthetically, during this study we serendipitously found a new family of eukaryotic CCOs, the ACOL family. This family has several members in animal genomes but lacks the -PDPCK- motif. These findings underscore the importance of the -PDPCK- motif in this important family of proteins. Three manuscripts describing these results were published during this reporting period, and further studies are underway. b) During the last reporting period we published a manuscript describing a knock-in mouse model of a presumptive dominant-acting human RPE65 mutation, c.1430A>G, p.D477G mutation that has been reported to cause autosomal dominant retinitis pigmentosa (adRP). This dominant mutation is unique because all other human RPE65 mutation are recessively inherited. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. 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. We demonstrated that a splicing defect is associated with c.1430G pathogenesis. To extend this study currently we are examining the effect of this mutation on splicing in RPE cells derived from induced pluripotent stem cells (iPSCs) from patients affected with the RPE65 c.1430A>G point mutation. In addition, we will compare these with RPE cells derived from introduction of the point mutation via CRISPR/Cas 9 in isogenic iPSCs. These studies are ongoing. c) 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. These studies are ongoing. d) We began a project in May 2020 to study aspects of the mechanisms of translational pausing in SARS-CoV-2 spike (S) protein (under the NIH Intramural Targeted Anti-COVID-19 program). We seek to analyze the translation of native S protein, which we predict to be complex process and a possible avenue for therapeutic intervention. S protein is a viral surface protein by which SARS-CoV-2 gains entry into human cells. SARS-CoV-2 S protein has acquired a novel four amino acid insert -PRRA-, forming a new furin cleavage site and new glycosylation sites. We found that this novel insert (unique to SARS-CoV-2) is encoded by an RNA sequence that is extremely C:G-rich and is likely to form a very strong translation pausing site with properties similar to premature stop codons. However, current studies in the literature on expression of S protein do not analyze the original native cDNA sequence and instead use modified optimized sequences to enhance S protein expression. Based on bioinformatics data and current literature, we hypothesize that the expression of native S protein will be strongly affected by the novel pausing site. Research plans include: i) determining the effect of novel predicted pausing site on expression of native S protein in HEK293F cells; ii) determining the effect of novel predicted pausing site on expression of native S protein in lentiviral pseudovirions in cells expressing different endogenous cell receptors for S protein; and iii) investigating how small molecules could affect the pausing site mechanism and inhibit production of S protein. It is anticipated that these studies will provide insights into native S protein biochemistry and potentially open new strategies for prevention/mitigation of COVID-19. These studies are ongoing.