Research Project
2224663
Lipoxygenases are lipid-peroxidating enzymes that have been implicated in a variety of inflammatory diseases including bronchial asthma, arthritis and psoriasis. Lipoxygenases are designated 5-, 12- or 15-lipoxygenase based on their ability to oxygenate arachidonic acid at carbon 5, 12, or 15. Recently, 15-lipoxygenase has been shown to be induced in the inflammatory cells of human atherosclerotic lesions. Furthermore, the enzyme is colocalized with oxidized low density lipoprotein (LDL) in these lesions. This is significant in that 15-lipoxygenase is capable of oxidizing low density lipoprotein to its atherogenic form and the oxidation of LDL is believed to be a .key step in atherogenesis. Therefore, understanding the molecular basis of the lipoxygenase reaction is important to future therapeutic interventions in a host of inflammatory conditions. Several of the lipoxygenases have been purified and characterized and multiple cDNAs have been isolated. Lipoxygenases have been shown to be iron-bound metalloproteins. Despite these advances, little is known about the structural features of 15-lipoxygenase that are responsible for catalytic function. In addition, no three-dimensional structure of any lipoxygenase has been determined. This proposal is designed to study the structural biology of human and rabbit 15-lipoxygenase. The human enzyme will be overexpressed and purified using recombinant DNA techniques. The rabbit enzyme has been shown to be readily obtainable in pure form. Mutations in the cDNA for human 15-lipoxygenase will be made to evaluate the structural basis for substrate specificity and iron-binding. In addition, newly developed active-site probes will be used on pure protein to identify potential catalytic residues. The contribution of these residues to catalytic function will then be assessed using site-directed mutagenesis. Finally, efforts to define the three-dimensional structure of rabbit or human 15-lipoxygenase will continue. This structural biological approach to the study of 15-lipoxygenase can potentially increase our understanding of the molecular basis of the enzyme's action and suggest novel approaches to pharmacological intervention.
