title: Molecular mechanisms of the cytotoxicity of human α-lactalbumin made lethal to tumor cells (HAMLET) and other protein-oleic acid complexes.
abstract: Although HAMLET (human α-lactalbumin made lethal to tumor cells), a complex formed by human α-lactalbumin and oleic acid, has a unique apoptotic activity for the selective killing of tumor cells, the molecular mechanisms of expression of the HAMLET activity are not well understood. Therefore, we studied the molecular properties of HAMLET and its goat counterpart, GAMLET (goat α-lactalbumin made lethal to tumor cells), by pulse field gradient NMR and 920-MHz two-dimensional NMR techniques. We also examined the expression of HAMLET-like activities of complexes between oleic acid and other proteins that form a stable molten globule state. We observed that both HAMLET and GAMLET at pH 7.5 were heterogeneous, composed of the native protein, the monomeric molten globule-like state, and the oligomeric species. At pH 2.0 and 50 °C, HAMLET and GAMLET appeared in the monomeric state, and we identified the oleic acid-binding site in the complexes by two-dimensional NMR. Rather surprisingly, the binding site thus identified was markedly different between HAMLET and GAMLET. Furthermore, canine milk lysozyme, apo-myoglobin, and β2-microglobulin all formed the HAMLET-like complex with the anti-tumor activity, when the protein was treated with oleic acid under conditions in which their molten globule states were stable. From these results, we conclude that the protein portion of HAMLET, GAMLET, and the other HAMLET-like protein-oleic acid complexes is not the origin of their cytotoxicity to tumor cells and that the protein portion of these complexes plays a role in the delivery of cytotoxic oleic acid molecules into tumor cells across the cell membrane.
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita-10 Nishi-8, Kita-ku, Sapporo 060-0810, Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.affiliation: Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1, Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Ryusho
authors.initials: R
authors.lastname: Kariya
authors.affiliation: Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, 2-2-1, Honjo, Chuo-ku, Kumamoto 860-0811, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Okada
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita-10 Nishi-8, Kita-ku, Sapporo 060-0810, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita-10 Nishi-8, Kita-ku, Sapporo 060-0810, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan; Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan; Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Electronic address: kuwajima@ims.ac.jp.
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004354:Drug Screening Assays, Antitumor; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007768:Lactalbumin / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology; D009829:Oleic Acids / Q000494:pharmacology*; D011485:Protein Binding
chemical_list: D000970:Antineoplastic Agents; C506422:HAMLET complex, human; D009829:Oleic Acids; D007768:Lactalbumin
keywords: Alpha-Lactalbumin; Apoptosis; Biophysics; Fatty Acid; HAMLET; Molten Globule; Nuclear Magnetic Resonance; Protein Folding; Protein-Drug Interactions; Tumor Therapy