Zeng_2025_J.Vis.Exp__

We developed a novel, cost-effective, and highly sensitive assay for assessing liposomal membrane permeability based on ligand substitution reactions involving ligands encapsulated in the inner volume of liposomes, which we combined with a hydrolytic activity assay (using coenzyme A acylation) and molecular modeling simulation to investigate whether phosphates of the inositol ring in phosphatidylinositol protect membranes from the damaging effects of aberrantly expressed enzymes. Aberrant expression of phospholipases A2 (PLA2) leads to demyelination of axons that triggers the development of multiple sclerosis and Alzheimer's disease. To mimic the effects of aberrantly expressed PLA2, we used the basic subunit of viper venom PLA2, HDP-2P. The HDP-2P was tested on phosphatidylcholine (PC) liposomes enriched with phosphatidylinositol, phosphatidylinositol-4-phosphate (PI-4-P), or phosphatidylinositol-4,5-diphosphate (PI-4,5-P2). While HDP-2P showed minimal hydrolytic activity and only slightly increased permeability in PC liposomes, enrichment of PC liposomes with PI enhanced both hydrolysis and permeability. Remarkably, PI-4-P and PI-4,5-P2 dramatically inhibited HDP-2P's hydrolytic activity and completely reduced permeability. Molecular modeling simulations revealed that phosphates of the inositol ring in phosphatidylinositol sterically block phospholipid access to HDP-2P's active site, preventing hydrolysis. Both in vitro and in silico data suggest that phosphorylated phosphatidylinositols are associated with reduced PLA2 activity, indicating a potential mechanism for mitigating excessive PLA2 activity -- presumably as a means of protecting cell membranes from degeneration.