The objective of this application is to study the biochemical properties and mechanisms of regulation of an arachidonoyl-hydrolyzing phospholipase A2 (PLA2) from the mouse macrophage cell line, RAW 264.7. The production of the bioactive lipid mediators, the eicosanoids and platelet-activating factor, is initiated by the activation of a PLA2 in inflammatory cells. Despite the important role of PLA2 in the production of inflammatory lipid mediators, little is known about the properties of this enzyme. A calcium requiring, high molecular weight PLA2 has been purified from the macrophage cell line, RAW 264.7. This enzyme exhibits unique properties, including specificity for sn-2 arachidonic acid, suggesting a role in lipid mediator production. The PLA2 also has properties similar to the amphitropic, calcium/phospholipid binding proteins, such as protein kinase C. The activity of the PLA2 is stimulated by anionic phospholipids and diacylglycerol; and anionic phospholipids decrease the calcium concentration required for full activity of the enzyme. In addition, the PLA2 is found in the cytosol, but exhibits calcium-dependent association with membrane. These properties may play a role in the regulation of PLA2 activity in vivo.
One specific aim of this proposal, is to stimulate the RAW 264.7 macrophages and to determine if there is an increase in activity of the arachidonoyl-hydrolyzing PLA2, whether it translocates to membrane and whether these events correlate with changes in the intracellular calcium concentration. The role of specific membrane lipids in mediating membrane binding of the PLA2 and modulating the substrate specificity of the enzyme will be investigated in vitro using model membranes. The PLA2 is hypothesized to be regulated by postranslational modification, such as phosphorylation, acylation or proteolytic processing, that play a role in modulating PLA2 activity and binding to the membrane. These processes will be studied in vitro and in vivo. A priority is to obtain a PLA2 antibody for studies of posttranslational modification of the enzyme and translocation to specific membranes.
The final aim i s to determine the sequence of the PLA2 cDNA, in order to deduce the amino acid sequence of the enzyme. This will allow elucidation of important structural domains and homology comparisons to the low molecular weight PLA2 enzymes and to the amphitropic, calcium/phospholipid binding proteins. Elucidating the properties and mechanisms of regulation of enzymes involved in lipid mediator production is critical for a better understanding of potential methods for controlling the inflammatory process.
| Mould, Kara J; Barthel, Lea; Mohning, Michael P et al. (2017) Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury. Am J Respir Cell Mol Biol 57:294-306 |
| Gibbings, Sophie L; Thomas, Stacey M; Atif, Shaikh M et al. (2017) Three Unique Interstitial Macrophages in the Murine Lung at Steady State. Am J Respir Cell Mol Biol 57:66-76 |
| Zemski Berry, Karin A; Murphy, Robert C; Kosmider, Beata et al. (2017) Lipidomic characterization and localization of phospholipids in the human lung. J Lipid Res 58:926-933 |
| Frasch, S Courtney; McNamee, Eóin N; Kominsky, Douglas et al. (2016) G2A Signaling Dampens Colitic Inflammation via Production of IFN-?. J Immunol 197:1425-34 |
| Janssen, William J; Bratton, Donna L; Jakubzick, Claudia V et al. (2016) Myeloid Cell Turnover and Clearance. Microbiol Spectr 4: |
| Yun, Bogeon; Lee, HeeJung; Jayaraja, Sabarirajan et al. (2016) Prostaglandins from Cytosolic Phospholipase A2?/Cyclooxygenase-1 Pathway and Mitogen-activated Protein Kinases Regulate Gene Expression in Candida albicans-infected Macrophages. J Biol Chem 291:7070-86 |
| Desch, A Nicole; Gibbings, Sophie L; Goyal, Rajni et al. (2016) Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes. Am J Respir Crit Care Med 193:614-26 |
| Jayaraja, Sabarirajan; Dakhama, Azzeddine; Yun, Bogeon et al. (2016) Cytosolic phospholipase A2 contributes to innate immune defense against Candida albicans lung infection. BMC Immunol 17:27 |
| Kandasamy, Pitchaimani; Numata, Mari; Berry, Karin Zemski et al. (2016) Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling. J Lipid Res 57:993-1005 |
| Zemski Berry, Karin A; Murphy, Robert C (2016) Phospholipid Ozonation Products Activate the 5-Lipoxygenase Pathway in Macrophages. Chem Res Toxicol 29:1355-64 |
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