Eicosanoids mediate a wide variety of physiological processes including inflammation. These lipid mediators are oxygenated metabolites of arachidonic acid and other poly- unsaturated fatty acids. Arachidonic acid levels in cells are tightly controlled by the release of this fatty acid from cell membranes by the action of one or more phospholipases A2. We are studying a family of phospholipases A2 called cytosolic phospholipases A2 (cPLA2s) including cPLA2?, which has been shown to catalyze arachidonic acid release from cellular membranes. We propose to continue our studies of the regulation of cPLA2? in mammalian cells by phosphorylation and by specific protein-membrane lipid interactions. The mammalian genome contains 6 cPLA2s isoforms. We will prepare recombinant forms of all of these proteins so that we can study the specificity of designed inhibitors toward the full set of cPLA2s. We will also use structure-guided design to prepare inhibitors that can distinguish between the 1 and 6 cPLA2 isoforms since these are the two cPLA2s that most likely play a role in arachidonic acid release. We will also study the role of cPLA2s and secreted phospholipases A2 (sPLA2s) in lipid mediator production from human platelets stimulated with a variety of agonists. Finally, we will further develop a new method for high sensitivity, multiplex analysis of eicosanoids that will be very useful in the inflammation field for studying eicosanoid levels in precious biological samples.
The relevance of our work is to discover the molecular basis for eicosanoid production in mammalian cells and tissues. Eicosanoids are lipid mediators that mediate inflammation including asthma and arthritis. The enzymes involved in generating free arachidonic acid, the precursor of the eicosanoids, are being identified, and their regulation is being studied.
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