Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of a few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis induce the activation of the ESCRT-III-dependent membrane repair machinery, which counterbalances the kinetics of cell death and modulates the immunological signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained increase of cytosolic Ca2+ prior to plasma membrane rupture is a common feature of regulated types of necrosis and position ESCRT-III activation as a general protective mechanism in these lytic cell death pathways.

Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it has been shown that TNMs recognize 3β-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of sterol binding at a molecular level has not been determined. In this study, to gain insight into the mechanism of sterol recognition of TNM in lipid bilayers, surface plasmon resonance (SPR) experiments and solid-state deuterium nuclear magnetic resonance ((2)H NMR) measurements were performed on theonellamide A (TNM-A). SPR results revealed that the incorporation of 10 mol % cholesterol or ergosterol into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes significantly enhances the affinity of the peptide for the membrane, particularly in the initial binding to the membrane surface. These findings, together with the fact that binding of TNM-A to epicholesterol (3α-cholesterol)-containing liposomes and pure POPC liposomes was comparably weak, confirmed the preference of the peptide for the 3β-hydroxysterol-containing membranes. To further establish the formation of the complex of TNM-A with 3β-hydroxysterols in lipid bilayers, solid-state (2)H NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol. The (2)H NMR spectra showed that TNM-A significantly inhibits the fast rotational motion of cholesterol and ergosterol, but not epicholesterol, therefore verifying the direct complexation between TNM-A and 3β-hydroxysterols in lipid bilayers. This study demonstrates that TNM-A directly recognizes the 3β-OH moiety of sterols, which greatly facilitates its binding to bilayer membranes.

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. Although AM3 permeabilizes phospholipid membranes only in the presence of sterol, the detailed molecular basis by which AM3 recognizes sterols in membranes remains unknown. Here, we investigated the molecular interaction between sterols and AM3 in membranes from the viewpoint of stereospecific molecular recognition using ergosterol, cholesterol, and epicholesterol, which is the 3-OH epimer of cholesterol. Dye leakage assays, surface plasmon resonance experiments, (2)H and (31)P NMR measurements, and microscopic observations revealed that AM3 directly interacts with membrane sterols through the strict molecular recognition of the stereochemistry of the sterol 3-OH group. The direct interaction enhances the membrane binding efficiency of AM3, which subsequently permeabilizes membranes without altering membrane integrity.

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