Apoptosis is the physiological process of cell death responsible for the maintenance of normal tissue homeostasis. Deregulation of this process may underlie the pathogenesis of a variety of diseases including cancer, AIDS, autoimmune disorders and neurodegenerative diseases. Thus, the ability to pharmacologically modulate apoptosis in a positive or negative fashion may have therapeutic value. However, development of effective drugs is hampered by a poor understanding of the mechanism of apoptosis. This proposal is based on this laboratory's discovery of a novel apoptotic protease of MW 24kD (AP24) that functions in apoptosis to activate DNA fragmentation. The activity of this protease is controlled, at least in part, by a novel serpin-like inhibitor (AP24i) also discovered in this laboratory. This study proposes to clone and express the gene for AP24. AP24i will be purified and the amino sequence determined. The mechanism of action of these two proteins will be analyzed in the U937 histiocytic lymphoma model system of apoptosis induced by tumor necrosis factor alpha or UV light. Experiments will determine how AP24 is activated, how AP24i is inactivated and analyze mechanisms leading to DNA fragmentation. The role of sphingomyelinase in activation of AP24 and of oxygen free radicals in destruction of AP24i will be evaluated. Endonucleases activated in isolated nuclei by AP24 will be characterized. The generality of these findings will be explored in other model systems of apoptosis using a variety of normal and transformed lines responding to diverse apoptotic stimuli. This investigation should provide novel insight into the biochemical pathway of apoptosis and the potential for therapeutic intervention through modulation of AP24 or AP24i activity.
