This project will evaluate the properties and physiological role of a novel phospholipase A{2} (aiPLA{2}) that is one of the enzymatic activities of the protein called peroxiredoxin 6. We have isolated the protein from rat and bovine lungs, have identified the full length cDNA for the human, rat, mouse and bovine enzyme, have generated recombinant protein, and have developed a panel of antibodies that are effective for western blotting, immunoprecipitation and immunocytochemistry. aiPLA{2} is Ca[++]-independent, shows maximal activity at pH 4, and represents a lysosomal-type PLA{2}. During the present period of grant support, we have shown that aiPLA{2}: 1) plays a major role in degradation of internalized alveolar DPPC;2) provides substrate for the reacylation pathway of DPPC synthesis;3) activity is regulated through protein-protein interactions with SP-A;4) is localized in lung to lamellar bodies and lysosomes;5) is activated by phosphorylation;and 6) its expression is developmentally regulated and hormonally responsive.
Specific Aim 1 will study models of under- and over-expression of aiPLA{2} (knockout and transgenic mice) in order to evaluate the physiologic role of the enzyme in DPPC degradation and synthesis using isolated cells, the isolated perfused lung, and in vivo studies. Time-related (aging) changes in lung phospholipid content will be determined.
Specific Aim 2 will evaluate the structure-function relationships for aiPLA{2} activity utilizing site directed mutagenesis and biophysical techniques to determine the role of the proposed catalytic triad, specificity of the enzyme for phospholipid substrates, and identification of the binding sites for its interaction with SP-A. X-ray crystallography will be utilized to provide additional insights into the spatial interactions of enzyme, substrate, and inhibitors.
Specific Aim 3 will evaluate the targeting motifs responsible for the subcellular localization of the enzyme utilizing deletion mutants. The subcellular localization determines the phospholipase (lysosomal) vs. peroxidase (cytoplasmic) activities of this protein.
Specific Aim 4 will investigate the regulation of Prdx6 activity with emphasis on the role of protein phosphorylation. A focus will be on the MAP kinase pathway and the specific phosphorylation sites on the protein. These studies will provide important insights into the role and regulation of a novel enzyme of major importance to lung surfactant metabolism and will provide the basis for further in depth characterization of the role of lysosomal PLA{2} activity in disease pathogenesis.
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