α-Lactalbumin, Engineered to be Non-Native and Inactive, Kills Tumor Cells When in Complex with Oleic Acid: A New Biological Function Resulting from Partial Unfolding

Abstract

In addition to its function as an intermediate in nitrogen metabolism, the small molecule urea plays an important role in the homeostasis of osmolarity and fluid volume in many organisms, including mammals, which concentrate urea in the kidney to produce the osmotic gradient necessary for water re-absorption. Because urea is highly polar and consequently poorly permeable through lipid bilayers, specialized urea transporters have evolved to increase its rate of diffusion across cell membranes. To better understand how urea transporters achieve the rapid and selective transport of urea, we have solved the 2.3 A ˚structure of a urea transporter from the bacterium Desulfovibrio vulgaris (dvUT), which has significant homology to mammalian urea transporters. The dvUT fold contains two homologous domains related by a two-fold pseudosymmetry axis perpendicular to the plane of the membrane. Each protomer contains a continuous membrane-spanning pore, suggesting that the protein operates by a channel-like rather than transporter-like mechanism. The constricted selectivity filter at the center of pore can accommodate dehydrated urea molecules passing in single file. Urea is stabilized by backbone and side chain oxygen atoms that provide continuous coordination as it progresses through the filter, and by well-positioned a-helix dipoles. We are now using a variety of functional assays to probe the physical and chemical interactions involved in the transport mechanism, as well as interactions between dvUT and various high-affinity UT blockers.

α-Lactalbumin:Oleic Acid Complex Spontaneously Delivers Oleic Acid to Artificial and Erythrocyte Membranes

“…We prepared BLAOA, a complex similar to BAMLET, using a simple incubation of BLA in the presence of OA under mild conditions favoring partial unfolding (Ca 2+ -depletion and 37 ºC) 22 . This approach provides the opportunity to A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 4 form BLAOA with varying amounts of OA included in the complex. Our subsequent analysis showed that both the complex ability to induce leakage and cell death correlated well with the amount of OA bound in the complex.…”

“…The phenomenon of cytotoxic protein-oleic acid (OA) complexes has received continuous attention in recent years, in particular HAMLET (Human -Lactalbumin Made Lethal to Tumors) and its related complexes [1][2][3][4][5] . Such complexes are more generally known as proteinfatty acid (PFA) complexes and the focus these have received are partly due to their possible A C C E P T E D M A N U S C R I P T…”

Structure and function of human α‐lactalbumin made lethal to tumor cells (HAMLET)‐type complexes

“…The 1 H‐NMR spectrum of HAMLET exhibited broad peaks with poor chemical shift dispersion, indicating a protein in conformational exchange on the millisecond timescale. The NMR signals corresponding to oleic acid were detected in the spectrum and the signal was broader than oleic acid alone, suggesting that the fatty acid was integrated into the protein [2,31]. Recombinant wild‐type α‐lactalbumin, expressed in Escherichia coli , showed identical CD and ANS spectra as the native protein and was readily converted to HAMLET on an oleic acid‐conditioned column [2].…”

Effects of pH and protein conformation on in-solution complexation between bovine α-lactalbumin and oleic acid: Binding trend analysis by using SPR and ITC

“…From the fitting, the stoichiometry was determined to be ca. 5.2 and the dissociation constant (K d ) was ~2.5 x 10 -4 M. The stoichiometry value of 5.2 is in good agreement with 5.1 -5.4 for the HAMLET complex prepared by the original chromatographic method[4,10,30]. Meanwhile, the enthalpy change (ΔH) was -4.3 kJ/mol, the entropy change (T·ΔS) was 17.2 kJ/mol, and the Gibbs free-energy change was -21.5 kJ/mol.…”

“…Removing the bound calcium ion induces changes in the tertiary structure of α-LA [7][8][9] to form a stable folding intermediate; a molten globule in which rigid tertiary structure is lost but native-like secondary structure is maintained [10,11]. Also, it was reported that addition of 7.5 equivalent of oleic acid to bovine α-LA (pH 8.3, 5 mM EDTA) at 20℃ induces the conformational changes resembling those observed in the formation of molten globule [12].…”