HAMLET forms annular oligomers when deposited with phospholipid monolayers.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Human α-lactalbumin made lethal to tumor cells (HAMLET) exhibits potent anticancer activity.
• HAMLET's membrane affinity and cytotoxic effects are pH-dependent.
• Native bovine α-lactalbumin (BLA) is inactive, serving as a control.

Purpose of the Study:

• To investigate the structural basis of HAMLET's membrane interaction and anticancer activity.
• To explore the formation of specific protein structures (annular oligomers) upon membrane interaction.
• To correlate protein structure with membrane disruption and cytotoxic potential.

Main Methods:

• Atomic force microscopy (AFM) to visualize protein-induced membrane structures.
• Liposome content leakage assays to assess membrane permeability.
• Circular dichroism (CD) spectroscopy to study protein folding states.

Main Results:

• HAMLET forms distinct annular oligomers (AOs) on negatively charged lipid monolayers at neutral pH.
• BLA, BAMLET, and Peptide C also form AO-like structures, but at higher concentrations than HAMLET.
• Peptide A, which binds membranes only at acidic pH, did not form AOs.
• AO formation correlates with membrane integration and liposome leakage, suggesting a role in HAMLET's cytotoxicity.
• HAMLET's propensity to form intermediately folded states, influenced by oleic acid, is linked to AO formation.

Conclusions:

• Annular oligomer formation is associated with HAMLET's membrane-disrupting ability and anticancer activity.
• The capacity of HAMLET and related peptides to integrate into membranes at neutral pH correlates with AO formation.
• Intermediately folded states of HAMLET likely contribute to its ability to form AOs and exert cytotoxic effects.