EUROCORES EuroMEMBRANE_OXPL-Protein targets of oxidized phospholipids relevant to apoptotic signaling of acid sphingomyelinase

Our current studies in the framework of the SFB Lipotox aim at elucidating the toxicity mechanisms of oxidized phospholipids (oxPL) in macrophages. To understand the molecular basis of lipid-induced cell death, we investigate the uptake and stability of these lipids in cultured cells, and identify their primary targets on the proteome and transcriptome level. In addition, we analyze the intracellular signaling that is triggered by these compounds with emphasis on the apoptotic pathways associated with sphingolipid metabolism (Aim 3). For these experiments, established cell lines and primary cell preparations are used. The entire project is based on chemically well defined lipid oxidation products. Fluorescent analogs of oxPLs are being used as specific molecular tools for the identification of the primary molecular targets and the lipolytic activities responsible for oxPL degradation. In the framework of the EU project OXPHOS, we will characterize the interactions of the oxPLs with their target proteins in living cells and their physiological consequences as follows. 1. GFP-tagged proteins will be expressed in order to identify the intracellular localization of the protein targets that are identified by the functional proteomic studies in the SFB Lipotox (see the enclosed poster which was presented at the 2nd International Graz Symposium on Lipid and Membrane Biology: Focus on Lipotoxicity, March 13-15, 2008 Graz, Austria). 2. Fluorescence energy transfer experiments will be performed to determine the spatial proximity between the fluorescent lipid analogs and the GFP-tagged protein candidates. 3. Knock down experiments using siRNA should help identify the lipid-associated proteins that are essential for activation of acid sphingomyelinase, which is a key component of apoptotic signaling, and the cytotoxic effect of oxPL. In summary, the proposed project should lead to the identification of the primary signaling proteins that are involved in the toxicity of oxidized phospholipids in vascular cells.

Oxidative stress is an unwanted side effect of natural and vital oxidation processes in the body. This phenomenon may, on the one hand, be a consequence of disease and environmental stress and can, on the other hand, induce the development of diseases. Oxidative modifications of biomolecules as a consequence of exposure to so-called reactive oxygen species is causally related to the development and progress of atherosclerosis. This chronic disease which is promoted by an unhealthy lifestyle and dietary habit, increases the risk of heart attack and stroke which are responsible for most deaths in the Western civilized world. Atherosclerosis is a lipid-associated metabolic disease and the oxidative modification of lipids and proteins in the bloodstream, especially those transported in the so-called low-density lipoproteins (LDL), play a key role in atherogenesis. This effect is largely responsible for the observation that LDL and its components are not targeted to the peripheral cells, but accumulate in the cells of the arterial wall. This process is the onset of a fatal biochemical cascade finally leading to vessel occlusion. Our project was devoted to the toxicity of oxidized phospholipids (OxPl) in vascular cells. These bioactive lipids are generated upon oxidative modification of highly unsaturated phospholipids in LDL and cell membranes. Permanent exposure to OxPl impairs enzyme function, modifies lipid metabolism in vascular cells and leads besides other detrimental effects to cell death. In our project, we have used biochemical methods to identify the proteins that are directly targeted by the OxPl and, for the first time, directly visualized the respective interactions in live cells. For this purpose, the OxPl and their target proteins were labelled with fluorescent probes and observed with fluorescence microscopy on the single molecule level (cooperation with Martin Hof, Academy of Sciences, Prague, Czech Republik). We found that OxPl act on different subcellular levels. The results and the newly developed methods of this project have already led to follow up projects that aim at explaining the individual mechanisms of OxPl toxicity on a molecular level.