Oxidative stress generates oxidized phospholipids (OxPLs) that alter membrane structure and inflammatory lipid signaling, yet the underlying biophysical mechanisms remain poorly understood. Here, we examine how two structurally distinct truncated oxidized phosphatidylcholines (OxPCs), 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), remodel membrane lateral organization and regulate secretory phospholipase A₂ (sPLA₂) activity. Large unilamellar vesicles composed of sphingomyelin, cholesterol, and either monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or polyunsaturated 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) were used to reconstitute the liquid-ordered/liquid-disordered (L_o/L_d) phase coexistence characteristic of eukaryotic plasma membranes. Fluorescence spectroscopy revealed that OxPLs modulate lipid packing and nanodomain organization in a structure- and composition-dependent manner. POVPC promoted pronounced membrane ordering and L_o domain stabilization compared with PGPC, particularly in monounsaturated membranes with low cholesterol content. In contrast, PDPC-containing membranes, especially at elevated cholesterol, exhibited enhanced structural resilience to OxPL-induced perturbations. These biophysical changes were associated with distinct functional outcomes. Notably, the relationship between membrane structural parameters and sPLA₂ activity was not linear, indicating a decoupling between bulk membrane properties and enzymatic response. sPLA₂ activity was linked to membrane lateral organization: the size of L_o domains modulate hydrolysis by influencing the physicochemical properties of L_o/L_d interfaces, which may represent preferential sites for enzyme activation. Consistent with this, POVPC reduced sPLA₂ activity through stabilization of ordered domains at both low and high cholesterol, while PGPC enhanced hydrolysis at high cholesterol. Importantly, PDPC-containing membranes attenuated sPLA₂ activity and exhibited a protective effect against OxPC-induced enzymatic activation. Together, these findings identify membrane lateral organization as a key regulator of sPLA₂ function and provide mechanistic insight into how oxidative stress can differentially modulate inflammatory lipid signaling depending on membrane composition. This work highlights membrane organization as an active determinant of enzyme activity and a potential target in pathologies associated with oxidative stress, including atherosclerosis, neuroinflammation, and metabolic disease.

Petasin, a lipophilic sesquiterpene ester isolated from Petasites hybridus, exhibits well-documented anti-inflammatory and anti-allergic activities, although its molecular mechanisms of action remain incompletely understood. Here, we investigated its direct effects on membrane organization using biomimetic large unilamellar vesicles (LUVs) modeling resting and activated mast cell plasma membranes. The models comprised phosphatidylcholines (EggPC, PAPC), sphingomyelin (SM), ganglioside GD3, and cholesterol (Chol), with phosphatidylserine (PS) additionally incorporated into the activated membrane model. Simplified liquid-disordered (L_d) and liquid-ordered (L_o) lipid systems were used to resolve phase-dependent effects. Membrane properties were assessed by Laurdan generalized polarization, DPH fluorescence anisotropy, DPH-TEMPO fluorescence quenching, di-8-ANEPPS dipole potential measurements, and dynamic light scattering (DLS). Petasin exerted phase- and composition-dependent effects on membrane organization. It increased lipid order in relatively disordered membrane environments, while producing only minor changes in highly ordered L_o membranes, revealing a clear dependence of its effects on the initial lipid phase state. In heterogeneous mast cell membrane models exhibiting L_o/L_d phase coexistence, DPH-TEMPO quenching indicated a concentration-dependent increase in the estimated size of ordered lipid nanodomains. In parallel, petasin decreased membrane dipole potential in a concentration-dependent manner, demonstrating modulation of membrane interfacial electrostatic properties. These findings indicate coordinated modulation of lipid packing, lateral membrane organization, and interfacial electrostatics, with the membrane response influenced by lipid composition. DLS measurements showed no evidence of vesicle aggregation or major alterations in vesicle size distribution. Overall, these findings identify petasin as a membrane-active modulator capable of fine-tuning lipid order, lateral membrane organization, and interfacial electrostatics in a phase- and composition-dependent manner. Given the importance of membrane organization and lipid raft dynamics in FcεRI clustering and mast cell activation, these membrane effects may represent an additional biophysical mechanism of the anti-inflammatory and anti-allergic actions of petasin. More broadly, our findings highlight membrane physicochemical organization as a relevant factor in the regulation of mast cell function and provide a basis for further investigation of membrane-mediated mechanisms in allergic and mast cell-associated inflammatory responses.