) It is important to understand the process by which mammalian cells liberate arachidonic acid from membrane phospholipids for the biosynthesis of prostaglandins, leukotrienes, and other oxygenated arachidonic acids (eicosanoids) since these molecules play important roles in inflammation and other physiological responses. It is generally accepted that the glucocorticoid steroids exert their antiinflammatory properties, at least in part, by blocking the cell's ability to liberate arachidonate from membrane phospholipids. It now is virtually certain that arachidonate release is controlled by a collection of phospholipases A2, both secreted enzymes as well as a cytosolic form (cPLA2) that translocates the nuclear envelope in response to agonists that cause arachidonic acid liberation. There are several goals of the research described in the current proposal. cPLA2 is activated in cells in part by phosphorylation. Although we have identified two phosphoryltion sites on cPLA2 and have shown that phosphorylation at these sites enhances arachidonic acid liberation, we wish to examine cPLA2 phosphorylation in two additional mammalian cell lines since there are hints that cPLA2 might be regulated by novel types of phosphorylation in these cells. We have previously shown that cPLA2 translocates from the cytosol to the nuclear envelope in stimulated cells, and we will make attempts to understand the molecular basis of this membrane targeting. The x-ray crystal structure of human cPLA2 has been determined by Dessen and co-workers. Building on this structural information, we propose to make improvements to our first generation of cPLA2 inhibitors by designing more potent analogs. We also propose to use our recently developed technique for docking proteins onto membranes (based on spin labeled protein and electron paramagnetic resonance) to answer three questions about cPLA2 membrane binding: 1) How is its calcium-dependent membrane binding domain (C2 domain) bound to membranes?; 2) What protein surface constitutes the membrane binding element of the catalytic domain of cPLA2?; 3) How does the protein element that block access of the active site to substrate, the lid, re-position when cPLA2 binds to membranes?
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