Abstract
Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.
Highlights
HAMLET is a broadly tumoricidal complex of partially unfolded ␣-lactalbumin and oleic acid whose structural and functional contributions to HAMLET remain largely undefined
Oleate Is the Functional Cofactor in HAMLET—The protonation state of bound oleic acid in HAMLET was determined by natural abundance 13C Nuclear Magnetic Resonance (NMR) spectroscopy
An additional peak at 175 ppm, verified to be the carboxyl carbon of residual EDTA in the conversion process, was recorded. These results identify oleate as the lipid cofactor in the HAMLET complex
Summary
HAMLET is a broadly tumoricidal complex of partially unfolded ␣-lactalbumin and oleic acid whose structural and functional contributions to HAMLET remain largely undefined. Long-chain fatty acids are essential cellular components, serving as nutrients, membrane constituents, signaling molecules, and precursors for prostaglandins and other crucial bioactive substances [1] Examples of their effects include modifications of enzymatic function, gene expression, synaptic transmission, and metabolism [2,3,4,5]. HAMLET2 (human alpha-lactalbumin made lethal to tumor cells) is a complex of ␣-lactalbumin and oleic acid [13, 14] and the first member in a new family of complexes formed from partially unfolded proteins with fatty acids as integral constituents. Studies of complexes with high lipid content have recently suggested that unfolded proteins may function solely as “lipid carriers” and that the tumoricidal response is triggered by the long-chain fatty acids alone [28, 30]. Fatty acids may exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein
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