Leukotrienes are potent lipid mediators, generated by the enzyme 5-lipoxygenase (5LO), and released from inflammatory mast cells, eosinophils and macrophages. There is good evidence to support the role of leukotrienes in the proinflammatory events and pathogenesis of asthma, a common, chronic disorder which afflicts nearly ten percent of the U.S. population. In light of recent data showing the presence of this enzyme in the nucleus of certain inflammatory cells, associated with euchromatin, which are areas of active gene transcription, one hypothesis to be explored in this proposal is that there are novel, leukotriene-independent functions of this protein in the nucleus. An integrated plan will be implemented to define further the biochemistry and biology of 5LO and leukotrienes.
In Specific Aim 1, the ATP-binding domain of 5LO will be characterized by photoaffinity labeling and in vitro site-directed mutagenesis experiments in an effort to understand better the functional importance of the ATP cofactor.
In Specific Aim 2, the signals directing 5LO to the nucleus, the specific sites of translocation, and interaction with DNA will be studied in mast cells. The role of 5LO in transcriptional regulation, and phosphorylation status of the protein in IgE/antigen-activated cells will be examined.
In Specific Aim 3, the role of 5LO products in the development of airways hyperresponsiveness and the immune response will be investigated in mice with a targeted disruption of the 5LO gene using an allergic airway inflammation model that displays the hallmark traits of asthma. Since both of these parameters were significantly diminished in 5LO deficient mice in initial studies, a focus in this proposal will be reconstitution of the responses via bone marrow-derived precursor cells containing 5LO and recombinant adenoviral delivery. These studies should provide a means to elucidate the important leukotriene-dependent and independent functions of 5LO in allergic inflammation and inflammatory cells, as well as bring to light the interaction of ATP with 5LO.
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