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It has been shown that a folding variant of alpha-lactalbumin containing oleic acid (HAMLET: Human Alpha-lactalbumin Made LEthal to tumor cells) induces apoptosis in tumor cells whereas healthy cells remain resistant. Furthermore, the apoptotic activity of HAMLET seems related to fundamental physicochemical features, such as the ability to populate partially unfolded conformations, likely associated to a marginal folding/unfolding free-energy barrier. Here, we explore the possibility of using protein engineering to create HAMLET-like behavior in proteins other than alpha-lactalbumin. To this end, we have introduced in a suitable protein model system mutations that are expected to affect the thermodynamic folding barrier and we have probed the oleic-acid binding capability of the variants thus obtained. Finally, the tumoricidal activity of the resulting protein/oleic-acid complexes has been characterized.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-lactalbumin on the stationary phase in situ. Because native alpha-lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.