α‐Lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET ( h uman α ‐lactalbumin m ade le thal to t umor cells)

HAMLET ( h uman α ‐lactalbumin m ade le thal to t umor cells) is a complex of human α‐lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis‐like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α‐lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α‐lactalbumin is sufficient to induce cell death. We used the bovine α‐lactalbumin Ca 2+ site mutant D87A, which is unable to bind Ca 2+ , and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET‐like complex in the presence of oleic acid. BAMLET ( b ovine α ‐lactalbumin m ade le thal to t umor cells) and D87A‐BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca 2+ site, as HAMLET maintained a high affinity for Ca 2+ but D87A‐BAMLET was active with no Ca 2+ bound. We conclude that partial unfolding of α‐lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca 2+ ‐binding site is not required for conversion of α‐lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca 2+ site.