Using Liprotides to Deliver Cholesterol to the Plasma Membrane.

Cholesterol (chol) is important in all mammalian cells as a modulator of membrane fluidity. However, its low solubility is a challenge for controlled delivery to membranes. Here we introduce a new tool to deliver chol to membranes, namely, liprotides, i.e., protein-lipid complexes composed of a fatty acid core decorated with partially denatured protein. We focus on liprotides prepared by incubating Ca2+-depleted α-lactalbumin with oleic acid (OA) for 1 h at 20 °C (lip20) or 80 °C (lip80). The binding and membrane delivery properties of liprotides is compared to the widely chol transporter methyl-ß-cyclodextrin (mBCD). Both lip20 and lip80 increase the solubility of chol ~ 50% more than mBCD and deliver chol to membranes with comparable efficiency. Although OA is cytotoxic at high concentrations, its effects are counterbalanced by chol. Further, cytotoxicity is strongly reduced when OA is replaced by cis-palmitoleic acid or cis-vaccenic acid. This makes liprotides good tools to deliver chol to membranes and cells.

Dynamic content exchange between liprotides.

Liprotides are complexes composed of partially denatured proteins and fatty acids in which the fatty acids form a micelle-like core surrounded by a shell of proteins. Liprotides, composed of α-lactalbumin (aLA) and oleic acid (OA), are similar in components and cytotoxicity to the original HAMLET protein-fatty acid complex. Liprotides composed of aLA and OA kill tumor cells by transferring the OA component to, and thus destabilizing, the cell membrane. Here we investigate liprotides' dynamics of transfer of contents between themselves and membranes using the hydrophobic fluorescent probe pyrene. We find that pyrene incorporated into liprotides is exchanged between liprotides within the dead time of a stopped-flow instrument, while the transfer to membranes occurs within 20s. Transfer kinetics was not affected by the presence of the membrane stabilizing lipid cholesterol. Thus, transfer is a remarkably rapid process which illustrates liprotides' efficacy as transporters of hydrophobic compounds.

Liprotides kill cancer cells by disrupting the plasma membrane.

HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and oleic acid (OA) which kills transformed cells, while leaving fully differentiated cells largely unaffected. Other protein-lipid complexes show similar anti-cancer potential. We call such complexes liprotides. The cellular impact of liprotides, while intensely investigated, remains unresolved. To address this, we report on the cell-killing mechanisms of liprotides prepared by incubating aLA with OA for 1 h at 20 or 80 °C (lip20 and lip80, respectively). The liprotides showed similar cytotoxicity against MCF7 cells, though lip80 acts more slowly, possibly due to intermolecular disulphide bonds formed during preparation. Liprotides are known to increase the fluidity of a membrane and transfer OA to vesicles, prompting us to focus on the effect of liprotides on the cell membrane. Extracellular Ca2+ influx is important for activation of the plasma membrane repair system, and we found that removal of Ca2+ from the medium enhanced the liprotides' killing effect. Liprotide cytotoxicity was also increased by knockdown of Annexin A6 (ANXA6), a protein involved in plasma membrane repair. We conclude that MCF7 cells counteract liprotide-induced membrane permeabilization by activating their plasma membrane repair system, which is triggered by extracellular Ca2+ and involves ANXA6.

Liprotides made of α-lactalbumin and cis fatty acids form core-shell and multi-layer structures with a common membrane-targeting mechanism.

α-Lactalbumin (aLA) has been shown to form complexes with oleic acid (OA), which may target cancer cells. We recently showed that aLA and several other proteins all form protein-OA complexes called liprotides with a generic structure consisting of a micellar OA core surrounded by a shell of partially denatured protein. Here we report that a heat treatment and an alkaline treatment method both allow us to prepare liprotide complexes composed of aLA and a range of unsaturated fatty acids (FA), provided the FAs contain cis (but not trans) double bonds. All liprotides containing cis-FA form both small and large species, which all consist of partially denatured aLA, though the overall shape of the species differs. Small liprotides have a simple core-shell structure while the larger liprotides are multi-layered, i.e. they have an additional layer of both FA and aLA surrounding the outside of the core-shell structure. All liprotides can transfer their entire FA content to vesicles, releasing aLA as monomers and softening the lipid membrane. The more similar to OA, the more efficiently the different FAs induce hemolysis. We conclude that aLA can take up and transfer a wide variety of FA to membranes, provided they contain a cis-bond. This highlights liprotides as a general class of complexes where both protein and cis-FA component can be varied without departing from a generic (though sometimes multi-layered) core-shell structure.