Structural Characterization of More Potent Alternatives to HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

Abstract

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids?

“…Earlier, we have shown that OA binding shifts the distribution of oligomeric forms of hLA, bovine α‐lactalbumin, bovine β‐lactoglobulin, and PA toward higher order oligomers . Since formation of the cytotoxic oligomers of IDPs plays an important role in pathogenesis of some human diseases, we have examined the OA/LA‐induced changes in the quaternary structure of the chosen proteins.…”

α-Lactalbumin, Amazing Calcium-Binding Protein

“…Curiously, the aforementioned complexes of bovine α-LA, bovine β-lactoglobulin, and pike parvalbumin with oleic acid were more potent against various tumor cells and strains of Streptococcus pneumonia than the original HAMLET complex. Subsequent biophysical analysis revealed that although the HAMLET-like complexes were characterized by marked structural differences, all of them exhibited a common feature, where the association of proteins with oleic acid resulted in a shift of the distribution of their oligomeric forms toward higher-order oligomers [ 297 ]. In addition to these proteins, lactoferrin also can form HAMLET-like complexes with oleic acid, which induce apoptosis in tumor cells through both death receptor- and mitochondrial-mediated pathways [ 298 ].…”

How Human Alpha-lactalbumin Recognize DNA and RNA

“…UDG "melts" ds d(pN) [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] partially and contacts with both chains of such relatively short ODNs almost independently [37,50]. In contrast, DNA polymerases [30,31], AP endonuclease [33], Topo I [39,40], 8-oxoguanine-DNA glycosylases [34,38] interact with both basepaired DNA strands.…”

“…This cofactor was identified as a specific fatty oleic acid [9]. It has been found that α-LA may be not the main cytotoxic component of its complexes with oleic acid, but the acid is more important [10,11].…”