Asthma prevalence is on the rise in the United States and other industrialized nations. Leukotrienes are prime perpetrators of the insidious properties of this disease, which include airway narrowing, hyperresponsiveness and inflammation. The proposed studies are geared towards a better understanding of the enzyme 5-lipoxygenase (5L0), which catalyzes the first step, in the formation of leukotrienes and how these compounds exert their actions through specific receptors. 5LO is predicted to be a two-domain protein consisting of a N-terminal beta-barrel domain of unknown function and a C-terminal non-home iron-containing catalytic domain, based on the crystal structures of other lipoxygenases. One hypothesis to be tested in this proposal is that the predicted N-terminal beta-barrel domain of 5LO binds calcium, interacts with membranes and is essential for 5LO translocation and activation. Thus, in Specific Aim 1 the 5LO beta-barrel will be purified and analyzed for calcium binding and phospholipid and membrane interactions by a variety of biochemical techniques. The crystal structure will be elucidated.
In Specific Aim 2 the role of the beta-barrel in 5LO translocation and intracellular localization in mast cells and trafficking of the enzyme will be functionally correlated with leukotriene biosynthesis. Functional analogies with the C2 domain of cytosolic phospholipase A2 will be sought. Green fluorescent protein-lipoxygenase chimera localizations will be followed in real time. The cysteinyl leukotriene receptor (CysLT1) that mediates leukotriene action in airways and its gene will be characterized in Specific Aim 3. Finally, the consequences of leukotriene receptor dysregulation will be examined in transgenic mice overexpressing CysLT1 in airways in Specific Aim 4. The mice will be investigated for eosinophil recruitment, mucus production and alterations in airway physiology in allergic airway inflammation models that mimic the asthmatic state. Current clinically relevant leukotriene receptor antagonists will be evaluated for inverse agonist properties in the CysLT1 overexpressing mice. These studies will furnish a comprehensive picture of the structure/function of the key enzyme in leukotriene formation and provide an important animal model for leukotriene-based allergic asthma. New insights into the pathophysiologic sequelae of leukotrienes in asthma and ultimately better treatments of this disease will be an outcome of these studies.
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