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.

Heme proteins which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the ″hemoglobin (Hb) superfamily″. The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct 2-on-2 α-helical globin fold. The truncated Hb (SynHb) from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the non-axial His 117 and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date and His117 is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a non-axial ″His″ into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, ESI-MS and MALDI TOF analyses confirmed the presence of covalent linkage in Mb Ile107His. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and autooxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb based blood substitute.

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.