HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Abstract

BackgroundCell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.Methodology/Principal FindingsWe show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLAall-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.Conclusions/SignificanceThe results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.

The cytotoxicity of fatty acid/α‐lactalbumin complexes depends on the amount and type of fatty acid

“…However, apart from these quite specific effects of HAMLET, there is also evidence of a more crude effect connected to membrane interactions and alterations. -LA binds to the membrane, but HAMLET causes loss of membrane integrity and leakage of lipid membranes [35] and ELOA, the cytotoxic complex between equine lysozyme and OA, has also been shown to induce loss of membrane integrity with ELOA accumulating at the cell membrane until the cells rupture and ELOA fills the cells [11,36].…”

The Alpha-Lactalbumin/Oleic Acid Complex and Its Cytotoxic Activity

“…In addition, it has been observed that -LA is able to rapidly insert itself into the lipid bilayer at pH 2 [75]. However, native human -LA is only inefficiently bound to and internalized by tumor cells, and does not translocate to the nucleus, nor influence tumor cell viability [3,5,68,69,76]. Even partial unfolding of human -LA has been shown to be insufficient for increased protein internalization into tumor cells [38].…”

“…Even partial unfolding of human -LA has been shown to be insufficient for increased protein internalization into tumor cells [38]. In addition, human -LA and oleic acid alone were unable to alter membrane structures, further indicating the necessity of interaction between unfolded -LA and an associated fatty acid for efficient internalization of the HAMLET complex in tumor cells [76].…”

“…A study of the interaction between HAMLET and natural or artificially generated, negatively charged, model lipid membranes has revealed that the interaction results in loss of cell membrane integrity, leakage of vesicular contents and morphological changes of the membranes [76]. Interestingly, HAMLET disturbs the integrity of the tested membranes, even under physiological conditions, as opposed to native-state -LA that only disrupts liposomes at acidic pH [77].…”

“…The ability to form oligomers is suggested to be a property of the -LA polypeptide chain, enhanced by the fatty acid component, which might be important for the cytotoxic activity of HAMLET [78]. It has been suggested that the degree to which HAMLET and other LA-OA-complexes are able to disturb the membrane integrity, depends on the lipid composition and physical characteristics of the membrane [74,76].…”