Characterisation of phospholipid-esterified eicosanoids generated by lipoxygenases. (360G-Wellcome-087596_Z_08_Z)

The proposed studies will address the hypothesis that PE-H(p)ETEs are transducers of LOX bioactivity in immune cells, through characterising the biology and fate of these lipids in platelets and monocytes/macrophages. Aim 1. Determine kinetics and mechanism of formation of PE-HETEs by purified recombinant LOX isoforms in vitro. The factors that regulate oxidation of PE versus arachidonate in intact cells are unknown. This aim will characterise PE oxidation mechanisms and kinetics using reco mbinant 12- and 15-LOXs in vitro. Aim 2. Characterise changes in the biological behaviour of PE by LOX oxidation. Introduction of a polar (O(O)H) group will change PE conformation, altering membrane physicochemical properties, including membrane thickness/phase. The ability of PE-H(p)ETEs to modulate membrane behaviour will be characterised by X-ray diffraction, using isolated cell membrane and purified lipid. Also, LOX regulation of PE dynamics in live cells will be determined using mass spectrometry and live cell imaging. Aim 3. Characterise PE-H(p)ETE metabolism and reactions of oxidised PE with cellular proteins via formation of covalent adducts or non-covalent interactions. Oxidized PE may interact with membrane proteins through a number of mechanisms. In this aim, decomposition of the hydroperoxy group of esterified HpETE including formation of protein adducts will be characterised.