Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells).
HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-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 alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-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 (bovine alpha-lactalbumin made lethal to tumor 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 alpha-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 alpha-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.
Svensson
Malin
M
Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
Fast
Jonas
J
Mossberg
Ann-Kristin
AK
Düringer
Caroline
C
Gustafsson
Lotta
L
Hallgren
Oskar
O
Brooks
Charles L
CL
Berliner
Lawrence
L
Linse
Sara
S
Svanborg
Catharina
C
D000596:Amino Acids / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007939:Leukemia L1210 / Q000473:pathology; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019301:Oleic Acid / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D000596:Amino Acids; D003470:Culture Media; D019301:Oleic Acid; D007768:Lactalbumin; D002118:Calcium