Modification of polyunsaturated phospholipids in cell membranes and lipoproteins by reactive oxygen species generates a plethora of lipid oxidation products. Oxidized phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of phosphatidylcholines containing arachidonic acid in position sn-2 of glycerol. Both lipids are characterized by a short polar fatty acyl chain in position sn-2 and a hydrophobic, long-chain, fatty acid in position sn-1 of glycerol. These compounds induce apoptosis in cultured vascular smooth muscle cells and macrophages. Their toxicity is associated with a very early increase in the activity of acid sphingomyelinase (acSMse) leading to the formation of the second messenger ceramide which transmits the apoptotic signal. It is still unclear how acSMse is activated by the oxidized phospholipids. This project aims at identifying the primary cellular targets of POVPC and PGPC and, among these candidates, the components that are associated with the function of this key signaling component mediating the apoptotic effects of both phospholipids.
The entire study will be based on chemically well defined lipid oxidation products, focusing on phosphatidylcholines containing homologous -aldehydo or -carboxyl fatty acids in position sn-2. We will screen a series of protein candidates for their capacity of binding or interacting with fluorescent analogs of oxidized phospholipids in cultured vascular smooth muscle cells and macrophages. From the lipid-associated polypeptides we will finally select those which are coupled to sphingomyelinase activation. Finally, the effect of the identified proteins on the capability of the host cells to undergo apoptosis under the influence of oxidized phospholipids. These experiments should finally lead to the identification of the primary signaling elements responsible for the apoptotic signaling of the oxidized phospholipids.