Center Affiliation and Expertise: Inflammation and Innate Immunity
Ongoing projects in the laboratory address several questions pertinent to the cell biology of neutrophil-mediated responses during inflammation and host response to infection. We have a longstanding interest in two important aspects of neutrophil biology, namely the NADPH-dependent oxidase and the granule hemeprotein myeloperoxidase (MPO), and are examining various aspects of each during acute inflammation. NADPH-dependent oxidase studies address two major questions. First, how does lipopolysaccharide, a component of most gram-negative bacteria, induce the neutrophil to become primed for subsequent activation? Detailed analysis of the subcellular localization and phosphorylation state of p47phox and p67phox are under study. Second, we are examining the role of the membrane-associated cytoskeleton in regulating and/or directing assembly of the NADPH-dependent oxidase. Current attention is focused on p57, a 57-kDa homologue of the protein coronin found in Dictyostelium, and its association with oxidase components and its recruitment to the nascent phagosome. Studies related to MPO include characterization of MPO biosynthesis and the identification of various genotypes of hereditary MPO deficiency. These include include characterization of the role of molecular chaperones calreticulin and calnexin as well as assessment of the various quality control mechanisms operating in the endoplasmic reticulum. We are searching for peptide motifs recognized by calreticulin and calnexin in their roles as chaperones. Studies are ongoing to define the mechanism by which heme is incorporated into MPO and the impact of heme insertion on the subsequent proteolytic processing and intracellular targeting of MPO. Studies of hereditary MPO deficiency include identification of the specific genotype and then characterization of the impact of that mutation on synthesis of MPO. For these studies we use transfected K562 cells to express mutant MPO cDNA and characterize the biosynthesis. Our findings from such analysis complement well those derived from studies of MPO biosynthesis in established myeloid cell lines.