Molecular Biology Of Outer Retina-specific Proteins

我们正在研究rpe的特异性机制，从调控和功能两个层面，研究RPE65的功能和调控，RPE65是视觉周期的关键视黄醇异构酶。目前的工作重点是建立RPE65催化的分子机制，以及它在光感受器发育和疾病中的调节和活性。我们也在研究视觉周期的类双维甲酸副产物（如A2E）对RPE溶酶体代谢的影响。此外，我们也在研究RPE65在RPE中表达的转录后调控。

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) 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. Here, we re-examined RPE65 palmitoylation, and its functional consequence in the visual cycle. We clearly demonstrate 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 C112 and C146. 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 show 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. In conclusion, 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 published during this reporting period. We are continuing this work by studying the biochemical and biophysical role of palmitoylation in RPE65. This work is in progress. b) In parallel with studies on palmitoylation in RPE65, we are studying 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 (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. This work is in progress. 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.1430A>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 displayed relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit 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 provide insights in the therapeutic strategy for human patients. A manuscript describing these results was published during this reporting period. We have been 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. In addition, this will be compared with RPE derived from introduction of the point mutation via CRISPR/Cas 9 in isogenic iPSCs. 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.