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-Aβ complexes are known to produce toxic partially reduced oxygen species (PROS), catalyze oxidation of neurotransmitters and have been associated with Alzheimer's disease (AD). Neuroglobin (Ngb) play a crucial neuroprotective role against oxidative damage, hypoxic injuries, stroke and apoptosis of neuronal cells. In this study, the interaction of heme-Aβ with apoNeuroglobin (apoNgb) has been investigated using a combination of spectroscopic techniques. Absorption and resonance Raman data confirm that apoNgb can uptake heme from heme-Aβ and constitute a six-coordinate low-spin ferric heme-active site identical to that of Ngb. ApoNgb can also uptake heme from reduced heme-Aβ resulting in the formation of ferrous Ngb. The rate of the heme transfer reaction has been found to be of the order of 10(6) M(-1) s(-1). The reaction is faster for oxidized heme-Aβ than the reduced form. The amount of PROS formation by heme-Aβ complexes has been found to diminish drastically after reaction with apoNgb. ApoNgb can also sequester ligand-bound heme from heme-Aβ, e.g., the CO-bound heme from heme-Aβ-CO complex resulting in the formation of Ngb-CO complex. Additionally, ApoNgb can sequester heme from self-assembled monolayer (SAM) of surface-bound heme-Aβ formed over Au surface. This heme sequestration by apoNgb from heme-Aβ not only diminishes heme-induced toxicity but more significantly it produces Ngb which has well-documented neuroprotective role and can thereby potentially reduce risks associated with AD.
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