w-Alkynyl Fatty Acids: Surrogates to Study Protein Adduction by Endogenously Generated Lipid Electrophiles
Beavers, William Norris
Polyunsaturated Fatty Acids (PUFAs) are oxidized both enzymatically and by autoxidation to many inflammatory signaling molecules, which have been detected in multiple disease states. Oxidized PUFAs can further react to form a series of electrophiles including unsaturated carbonyls and aldehydes. These electrophiles react with nucleophilic amino acid side chains of proteins forming adducts, potentially altering protein function. Many investigations of protein modification have been conducted following bolus addition of preformed electrophiles to purified proteins or cells, but questions persist as to the biological relevance of these studies. We developed ω-alkynyl linoleic acid (aLA) as a probe to study endogenous lipid electrophile generation and the resulting protein adduction. Electrophile-protein adducts containing an alkyne group can be enriched by addition of a biotin moiety via click chemistry followed by binding to then elution from streptavidin-coated beads. The addition of an alkynyl group to LA does not affect its autoxidation, oxygenation by lipoxygenases, transformation to ω-alkynyl arachidonic acid, or incorporation into lipid components of RAW264.7 macrophages. When electrophile formation is induced by lipopolysaccharide activation of aLA-incorporated RAW264.7 macrophages, we observed an increase in lipid electrophile adduction across the entire proteome. Stable Isotope Labeling of Amino acids in cell Culture was employed to quantify differences in lipid electrophile protein adduction before and after activation by an inflammatory stimulus. Our results show that during the inflammatory response, mitochondrial proteins are most heavily enriched for adduction. Targets of adduction include proteins important for oxidant defense and energy generation including superoxide dismutase 2 (Sod2), TCA cycle proteins, and proteins in complex V of the electron transport chain. We have measured a reduction in cellular ATP concentrations corresponding to adduction of electron transport chain proteins. Additionally, we have profiled the activity of Sod2, and see a significant reduction in activity correlating to an increase in adduction by lipid electrophiles. The activity of Sod2 is restored under conditions where lipid electrophile generation is inhibited. Modification of these proteins, which are vital for mitochondrial function, and the resulting change in activity, may contribute to disease initiation and propagation.