HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a complex of partially unfolded human alpha-lactalbumin and oleic acid that kills many different types of tumor cells and shows therapeutic efficacy in animal models and clinical studies. This thesis aims to (1) elucidate the structure of HAMLET and the exposure of biologically active domains, (2) define the contribution of lipids to the tumoricidal effect of HAMLET, (3) characterize the membranes response to HAMLET and the perturbation of membrane associated signaling cascades, (4) use proteomic screens to identify conserved features of HAMLET targets in tumor cells.
Elucidating the structure of HAMLET is important to understand its tumoricidal activity. Paper I present the first low-resolution solution structure of HAMLET, derived from small angle X-ray scattering data. In HAMLET, α-lactalbumin is partially unfolded, with an enlarged globular domain and an extended C-terminal conformation from L105 to L123. Synthetic globular or extended domain peptides triggered rapid ion fluxes in the presence of oleate, were internalized by tumor cells and caused rapid changes in cell morphology and tumor cell death with comparable efficiency as HAMLET. These findings demonstrate that the gain of tumoricidal activity in HAMLET is due to a loss of tertiary structure definition compared to native α-lactalbumin, which lacks such activity.
The contribution of the lipid to HAMLET’s tumoricidal activity has been debated. Paper II investigates the contribution of lipids to the tumoricidal effect of HAMLET. Deprotonated oleic acid (oleate) is identified as the functional cofactor in HAMLET and shown to contribute to some but not all of HAMLET’s cellular interactions. Partial effects on ion fluxes were observed in tumor cells but unlike HAMLET, oleate did not cause metabolic paralysis or cell death at concentrations relevant to HAMLET. Furthermore, oleate did not trigger cancer related gene expression. Cellular responses to oleic acid were weak or absent, suggesting that fatty acids exert some of their essential effects on host cells when in the deprotonated state. The results highlight the unique properties of the HAMLET complex compared to the lipid alone and suggest that the cellular effects of lipids may be modified in the context of a partially unfolded protein.
Membrane perturbations by HAMLET initiate cellular attack and death. Paper III identifies three critical molecular-level features for the conserved tumoricidal response. I. Rapid membrane perturbations in receptor-free model vesicles and tumor cells suggested that HAMLET-membrane interactions are receptor-independent. II. Formation of HAMLET-Ras membrane clusters in tumor cells and Ras inhibition provided a mechanism to activate a conserved cell death programs. III. Membrane responses were absent in differentiated cells, indicating tumor selectivity. The membrane perturbations might thus provide a physical means for HAMLET to excite membrane conformations serving as surrogate receptors for subsequent signal transduction, leading to cell death.
Paper IV examined the hypothesis that the apparent multitude of cellular targets reflects structural homology and that HAMLET targets epitopes shared by molecules critical for cell survival. By protoarray, HAMLET targets represent protein families critically involved in energy metabolism and cellular homeostasis including ATPases, kinases and small GTPases. In an in vitro kinase activity assay, about 70 % of kinases were inhibited by HAMLET. Broad kinase inhibition in HAMLET treated cells was confirmed by a phosphorylation antibody microarray, which identified kinases involved in cancer pathways. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells.
- Avdelningen för mikrobiologi, immunologi och glykobiologi - MIG
- Svanborg, Catharina, handledare
- Gruber, Gerhard, handledare, Extern person
- Hun Mok, Kenneth, handledare, Extern person
|Tilldelningsdatum||2014 sep. 12|
|Status||Published - 2014|
Place: Rune Grubb Salen, BMC, Sölvegatan 19, Lund
Name: Hunter, Tony
Affiliation: Salk Institute
- Mikrobiologi inom det medicinska området
- Immunologi inom det medicinska området