126 - Identification of Covalent Adducts Between Docosahexaenoic Acid and Its Oxidation Products with Thiols: The Potencial Significance of Thiol-Ene Reaction for Redox Signalling Processes

Abstract

Covalent coupling between unsaturated bonds in fatty acids and thiol groups in peptides and proteins can occur through a free radical (thiol-ene) and Michael addition reactions. Recently, we have observed that fatty acid induced-SOD1 aggregation in vitro depends on protein free thiol groups and the presence of least one unsaturated bond in the fatty acid structure. Docosahexaenoic acid (DHA; C22:6n-3) is highly enriched in the brain and its oxidation generates products bearing hydroperoxides, hydroxides and ketone moieties. The later, is known to form covalent adducts with thiol groups via Michael addition while unsaturated fatty acid can undergo a thiol-ene addition reaction. To better understand how DHA and its oxidation products could lead to thiol modifications we have investigated their reaction with thiols in a tri-peptide (glutathione, GSH) and a protein (Keap1) in vitro by LC-MS/MS and biotin-PEAC5-maleimide (BPM) assay. A new covalent adduct between DHA and glutathione (GS-DHA adduct, m/z 634.3168) was detected by LC-MS/MS. However, this adduct was only detected when incubated with AAPH, a free radical-generating azo compound, which suggeststhat GS-DHA is generated in presence of thiylradical. On the other hand, DHA derived hydroperoxides and ketones reacted with GSH generating a GS-oxo-DHA adduct (m/z 648.2960) while the hydroxides did not. To evaluate the reactivity of DHA and its oxidation products under more physiological conditions we evaluated their reactivity towards a thiol-rich protein, Keap1, the negative regulator of Nrf2. Incubations of DHA with Keap1 showed protein thiolate consumption as well as aggregation. Studies is now being conducted to characterize the presence of covalent DHA-thiol adducts in the protein and also to compare the reactivity between DHA and its oxidized derivatives. Overall, our data show that both non-oxidized and oxidized DHA derivatives can modify thiol groups in peptides and proteins, which may potentially affect redox signaling pathways.