HAMLET Forms Annular Oligomers When Deposited with Phospholipid Monolayers

Abstract

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes

“…It is thus strongly suggested that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-OA complexes probably works as a delivery carrier of the cytotoxic OA to tumor cells (30,38). It is becoming increasingly evident that not only the rigid native structures but also the flexible MG structures are biologically functional, and many of the functional MG states are related to translocations of proteins or hydrophobic ligands across membrane bilayers (56 -61), and the interaction of HAMLET with artificial and natural membranes has been reported (62)(63)(64). We thus conclude that the MG-like state in HAMLET, GAMLET, and the other related complexes plays a role in transferring the hydrophobic OA molecules into tumor cells across the cell membranes.…”

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

“…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]. Similarly, at physiological conditions HAMLET readily distorts lipid monolayers at low concentrations, while forming pore-like oligomeric structures resembling annular oligomers at higher concentrations [78]. 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].…”

“…Similarly, at physiological conditions HAMLET readily distorts lipid monolayers at low concentrations, while forming pore-like oligomeric structures resembling annular oligomers at higher concentrations [78]. 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].…”

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

“…HAMLET is able to form rings on negatively-charged membranes at neutral pH (68). This phenomenon is not thought to be involved with the compound's cytotoxic activity (68).…”