Research / Clinical Summary

Edward Dennis, PhD Professor, Chemistry & Biochemistry Tumor Growth, Invasion & Metastasis Program Contact by Email

Dr. Dennis' laboratory is focused on understanding the regulation of lipid second messengers and signal transduction processes and especially the role of various phospholipases in their generation. Special attention is paid to the cytosolic, secreted, and membrane-bound phospholipase A2s (PLA2) responsible for the control of prostaglandin and leukotriene biosynthesis in human and murine macrophage cell lines. The latter cells are of particular importance because they are extremely active in the biosynthesis of prostaglandins. These are produced from arachidonic acid released by PLA2 upon cell stimulation. Free arachidonic acid itself is implicated in signal transduction in a variety of cells as well as being a precursor of the eicosanoids including the prostaglandins and leukotrienes. Activation of PLA2 is associated with numerous inflammatory diseases. Current efforts are focused on a lipopolysaccharide (LPS) receptor for priming linked to a platelet-activating factor (PAF) receptor for activation of PLA2. Our goal is to characterize and elucidate the regulatory mechanisms of the phospholipases both in vitro and in the intact cell.

In their studies on the regulation and detailed mechanism of action of PLA2 at membrane and other lipid-water interfaces, their orientation is that of developing suitable in vitro systems for studying the detailed mechanism of action of membrane-bound enzymes and enzymes of lipid metabolism. PLA2s from various sources are currently receiving a great deal of attention because they represent the smallest (molecular weight 13,000) and perhaps the simplest enzymes of complex lipid metabolism known and are ideally suited for mechanistic studies. Their kinetic studies have led to the 'surface dilution model' for the action of the phospholipases on mixed micelles which entails the association of the enzyme with the lipid-water interface followed by Michaelis complex formation. PLA2s have several kinds of phospholipid binding sites including activator sites, interfacial sites, and catalytic sites. It is now important to define the precise role of the amino acid residues involved in these interactions as well as elucidating the conformation and physical state of the phospholipid in mixed micelles and membranes. The laboratory is now carrying out site-directed mutagenesis on various PLA2s derived from human genes as well as x-ray crystallographic, 2D-NMR, and computer modeling studies on these recombinant proteins.

Their laboratory also designs and synthesizes chemical inhibitors of phospholipase A2. Many different inhibitorclasses have been developed and studied including manoalide and manoalogue, thioether amide phospholipid analogues, and suicide inhibitory bifunctionally linked substrates (SIBLINKS). Extensive studies on lipocortins/annexins, which were thought to be physiologically relevant PLA2 inhibitors have also been performed. Studies are aimed at quantitating and understanding mechanisms of inhibition of phospholipases and other lipolytic enzymes. We are also studying synthetic oxidized phospholipids and their role in LDL scavenger receptor uptake, autoantibody formation, and apoptosis all of which are processes that may lead to cancer and disease. In summary, our laboratory utilizes organic synthetic approaches, enzyme kinetics, molecular biology, site-specific mutagenesis, cell and tissue culture, and high-resolution NMR techniques as well as traditional biochemical approaches in attacking phospholipase and membrane problems.

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