Problem: Leukotrienes (LTs) play important roles in normal host defense, and their over production is central to the pathogenesis of several inflammatory states, including asthma. The regulation of LT synthesis, as well as the dysregulation(s) that leads to their over production are poorly understood. Background: The enzyme 5-lipoxygenase (5-LO) catalyzes the initial steps in LT synthesis from arachidonic acid; thus delineating its regulation is essential to understanding the basis for LT synthesis and over production. In some cases, changes in LT synthesis correlate with changes in enzyme amount. However, there are numerous exceptions, indicating that other mechanisms for regulating LT synthesis must exist. We have recently reported that, in some cells, the enzyme 5-L0 is cytosolic, whereas in other cells it is intranuclear. Further, we have identified several conditions that can alter the compartmentation within a given cell type, suggesting that the movement of 5-LO into and out of the nucleus is a dynamic, regulated process. For example, we have demonstrated that 5-LO is in the cytosol of peripheral blood neutrophils, but that it quickly moves into the nucleus following either recruitment into sites of inflammation (in vivo) or adherence to artificial substrates (in vitro) (1). In both models of nuclear import of 5-LO, there is a significant (3- to 5-fold) increase in LT synthesis, compared to cells with cytosolic 5-LO. These results, and others, suggest that compartmentation of 5-LO may serve to regulate LT synthesis. Hypothesis: The differential compartmentation of 5-LO between the nucleus and the cytosol in resting cells is a regulated process; this, in turn, regulates the amount of LT synthesized upon cell activation.
Specific AIMS : (1) To characterize the regions within the 5-LO protein that are essential for (a) nuclear import and (b) nuclear export. (2) To evaluate the role of extracellular factors on 5-LO compartmentation. These factors includes extracellular matrices and endothelial and epithelial cell monolayers. (3) To define the role of protein phosphorylation in the regulation of 5-LO movement, specifically in (a) activation of import or export domains, both alone and in combination, and (b) activation by extracellular factors. (4) To determine how 5-LO localization, as regulated by the activity of nuclear import and export sequences, extracellular factors, and protein phosphorylation, affects the metabolism of arachidonic acid and, in particular, its conversion to LTs.
