Phospholipase A2 (PLA2) catalyzes the rate-limiting step in the production of eicosanoids that are potent mediators of inflammation. PLA2 has been implicated in various inflammatory diseases, including rheumatoid arthritis, asthma and septic shock. Thus, the elucidation of the mechanisms of functions and regulation of PLA2 is important for understanding the pathogenesis of inflammatory diseases. The primary objective of this research program is to determine the mechanisms of physiological regulation of two types of human PLA2S, secretory group V PLA2 (hVPLA2) and cytosolic PLA2-alpha (cPLA2), that have unique membrane binding and pro-inflammatory activities. The long-term objective is to develop therapeutic methods for treating PLA2-mediated inflammatory diseases.
Specific aims during this proposed period are: (1) to determine the molecular origin of unique membrane binding and pro-inflammatory activities of hVPLA2, (2) to study the mechanisms by which cPLA2 interacts with membranes and is activated, and finally (3) to determine the mechanisms whereby hVPLA2 and cPLA2 work in concert to produce eicosanoids in human neutrophils and eosinophils. The principal methodologies to be used include: (1) the site-directed mutagenesis and the over-expression of PLA2S, (2) kinetic and membrane-binding analyses of PLA2S using various model membranes developed in this laboratory and cell membranes, (3) surface plasmon resonance that allows for direct measurement of membrane association and dissociation rates, and finally (4) cell transfection of wild type and mutant PLA2S that are tagged with green fluorescence protein, followed by confocal imaging.
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