A07: Studying crosstalk dynamics of signaling complexes in extrinsic and intrinsic cell death pathways by MS-based proteomics

Project Leaders: Florian Stengel, Thomas Brunner

While members of the TNF family promote apoptosis via the extrinsic cell death pathway, chemotherapeutic drugs initiate the intrinsic, BCL-2 family-regulated, mitochondrial pathway. However, in some cell types there is considerable crosstalk between these two pathways. In particular, various tumor cells are resistant to both, TNF family ligands and chemotherapeutic drugs, yet the treatment of cells with sublethal doses of both stimuli results in synergistic induction of cell death. The underlying molecular processes of this synergy are far from being understood but are likely related to dynamic changes in the apoptosis-promoting signaling protein complex formation. In this study we will employ hepatocellular carcinoma cell lines to investigate the synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs at the level of TRAIL receptor complex formation at the plasma membrane, and BCL-2 interactome on the mitochondrial membrane. We will employ proteomics and structural mass spectrometry (MS), in particular crosslinking coupled to MS (XL-MS), to specify how synergistic signals affect composition and interactions of critical signaling molecules and molecular switches in these signaling complexes. While the project will assess how the relative abundances of known complex components will be altered by combined treatment with TRAIL and chemotherapy, it will also lead to the identification of potential novel regulatory proteins critical in the regulation of cell death/cell survival decisions by using MS-based workflows.