Phospholipase A2 (PLA2) which catalyzes the hydrolysis of membrane phospholipids has been implicated in various inflammatory diseases, including rheumatoid arthritis and septic shock. Long-term objectives of this research program are to elucidate the mechanisms by which pro-inflammatory PLA2 is regulated and to develop therapeutic methods for treating PLA2-mediated inflammatory diseases. To date the mainstay of PLA2 inhibition has been the active-site-directed inhibitors most of which lack either efficacy or specificity under physiological conditions. To develop an alternative approach to specific PLA2 inhibition, they have extensively studied the interactions of PLA2 with membranes (interfacial binding) and found that major classes of mammalian PLA2s have distinct interfacial binding modes. They propose to take advantage of these distinct membrane-binding properties in developing chemical and biological agents that specifically block the interfacial binding of two putative pro-inflammatory PLA2, secretory Class II PLA2 (hs-PLA2) and cytosolic PLA2 (cPLA2).
The Specific Aims during this proposed period are; (1) to determine the exact differences in interfacial binding mode between two secretory PLA2s; (2) to study the mechanism by which heparin regulates the in vitro and in vivo activity of hs-PLA2, identify the heparin-binding residues of hs-PLA2 and finally develop heparin derivatives with high hs-PLA2-inhibitory activity and low anti-coagulant activity; (3) to determine how differently cPLA2 interacts with membranes and how its interfacial binding is regulated by calcium and phosphorylation and identify interfacial binding residues of cPLA2. The principal methodologies they shall use include; (1) many recombinant DNA techniques for the site-directed mutagenesis and the over-expression of PLA2s; and (2) kinetic and membrane-binding analyses of PLA2 using the polymerized mixed liposome system developed in this laboratory which allows the systematic and independent analysis of the interfacial binding and catalytic steps.
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