HAMLET: Functional Properties and Therapeutic Potential

James Ho CS; Anna Rydström; Maria Trulsson; Johannes Bålfors; Petter Storm; Manoj Puthia; Aftab Nadeem; Catharina Svanborg


Future Oncol. 2012;8(10):1301-1313. 

In This Article

HAMLET: A Tumoricidal Complex of Partially Unfolded α-lactalbumin & Oleic Acid

The paradigm 'one gene, one protein and one function' was proposed by Tatum and Beadle;[16] however, since the completion of the human genome sequence in 2003, this paradigm has been widely debated.[17–19] Since the number of genes encoding proteins appears to be too small to fulfill the functional demands of complex organisms, additional mechanisms of functional diversification are being explored. While known structural modifications such as glycosylation and phosphorylation allow proteins to change their function and alternative splicing of mRNA transcripts may generate additional diversity, the HAMLET model suggests that partial unfolding enables proteins to generate new functional variants from a given polypeptide chain[10] (reviewed in[20]). When described, HAMLET was the first example of a protein that undergoes partial unfolding to attain a new function different from that of its native state. Importantly, HAMLET undergoes a partial unfolding and remains as such to fulfill its new biological function, clearly distinguishing proteins forming HAMLET and related protein–lipid complexes from the moonlighting proteins[21–23] and from the intrinsically unfolded proteins.[24] This mechanism also differs from amyloid formation, where unfolding is driven towards fibril formation, cytotoxicity for healthy tissues and the loss of function associated with numerous disease states.[25]

HAMLET is a complex of partially unfolded α-lactalbumin and oleic acid (C18:1:9 cis) (Figure 1).[10] The tumoricidal activity of HAMLET was discovered in casein, obtained after low pH precipitation of human milk[9] and isolated by ion exchange chromatography, when the active complex eluted as a sharp peak after high salt. Surprisingly, the major component of the eluate was the whey protein α-lactalbumin, which had not previously been identified in the casein fraction or associated with tumoricidal activity.[9,26] However, native α-lactalbumin was shown to lack tumoricidal activity and no post-translational modifications explaining the new activity were found. A loss of tertiary structure definition was detected by circular dichroism (CD) spectroscopy, however, this was accompanied by increased binding of the fluorescent hydrophobic dye, 8-anilinonaphtalene-1-sulfonic acid, suggesting that the low pH used for casein precipitation was driving a conformational change required for HAMLET formation.

Figure 1.

The two components of HAMLET with representation of the HAMLET model.
Native α-lactalbumin (Protein Data Bank ID: 1B9O) undergoes a depletion of calcium and binds to oleic acids to form HAMLET. A stoichiometric ratio of 1:5 (protein:fatty acid) is used for representation of the final HAMLET model. The structure is color coded, depicting a helix in red, the β-sheet in yellow and the random coil in green. Disulphide bonds are shown by stick representation.
The calcium ion is the blue sphere. Oleic acid structure is derived from Protein Data Bank ID: 1GNI.

The α-lactalbumin protein has been extensively used as a model of protein folding,[27–29] and partially unfolded states[27] have been studied by several techniques including hydrogen/deuterium exchange combined with NMR,[30,31] photochemically induced dynamic nuclear polarization NMR,[28] limited proteolysis[32] and mutational studies.[33] The crystal structure of human α-lactalbumin[34] reveals a large α-helical domain with three major α-helices and two short 310-helices. The small β-domain consists of a triple-stranded antiparallel β-sheet and a short 310-helix.[34] The protein is stabilized by four disulfide bonds[35] and a high-affinity calcium-binding site.[36]

While partial unfolding of α-lactalbumin is an essential feature of HAMLET formation, the structure of the HAMLET complex is not fully understood. In the near-UV range for the HAMLET CD spectrum a loss of tertiary structure definition has been detected,[10] and increased exposure of hydrophobic domains was suggested by a blue shift compared with the native protein, with increased 8-anilinonaphtalene-1-sulfonic acid binding in fluorescence spectroscopy. The 1H-NMR spectrum of HAMLET showed broader peaks as well as lower intensities of the upfield methyl proton peaks, indicating a less-ordered protein, confirming the near-UV CD result. Through hydrogen/deuterium exchange coupled with peptic digestion, a conformational state distinct from both the native and the Ca2+-depleted states was suggested.[37] Two constitutively unfolded α-lactalbumin mutants were used to prove this point. First, functional HAMLET complexes were produced from the D87 calcium-site mutant developed by Brooks and Berliner.[38] As this mutant does not bind calcium, it remains partially unfolded at solvent conditions used for the cell death assays. Second, a fully functional tumoricidal complex was readily formed from the Cys–Ala all-Ala mutant of α-lactalbumin.[39] This mutant lacks disulphide bonds with substitutions of all cysteines by alanines and remains non-native under all conditions. The studies of α-lactalbumin in HAMLET and of additional proteins (see below) illustrate how partial unfolding of proteins may be used as a successful route for functional diversification of proteins (Figure 2). Importantly, the constitutively unfolded mutants did not directly kill tumor cells, emphasizing the need for cofactors to form a functional HAMLET complex. The work has also been extended to address if peptides, rather than the entire protein, may possess tumoricidal activity. Tolin et al. have formed peptide–lipid complexes from peptic digests of α-lactalbumin, and have suggested that such peptides have tumoricidal activity, but only when bound to proper fatty acid cofactors.[40]

Figure 2.

Sequence variation among α-lactalbumins from different species.
Purified α-lactalbumins from these species readily form HAMLET-like complexes. However, acidification of milk samples from these species, analogous with casein precipitation of human milk, does not generate HAMLET-like complexes.
Reproduced with permission from [66].