| 1. |
- Fast, Jonas, et al.
(författare)
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Stability of HAMLET--A kinetically trapped {alpha}-lactalbumin oleic acid complex.
- 2005
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 14:2, s. 329-340
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Tidskriftsartikel (refereegranskat)abstract
- The stability toward thermal and urea denaturation was measured for HAMLET (human -lactalbumin made lethal to tumor cells) and -lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than -lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo -lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than -lactalbumin, although it is a complex of -lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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| 2. |
- Svensson, Malin, et al.
(författare)
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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).
- 2003
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 12:12, s. 2794-2804
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Tidskriftsartikel (refereegranskat)abstract
- HAMLET (human -lactalbumin made lethal to tumor cells) is a complex of human -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 -lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of -lactalbumin is sufficient to induce cell death. We used the bovine -lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, 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 -lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of -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 Ca2+-binding site is not required for conversion of -lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.
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| 3. |
- Svensson, Malin, et al.
(författare)
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Lipids as cofactors in protein folding: Stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells).
- 2003
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 12:12, s. 2805-2814
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo -lactalbumin and form HAMLET (human -lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein–fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo -lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid–protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.
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