Regulation of the internalization and intracellular transport of Tumor Necrosis-Factor Receptor 1 (TNF-R1) and its impact on signal transduction and biological outcomes of TNF

• Applicant: Professor Dr. Stefan Schütze
• Subject Area: Cell Biology
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 269124904

The proposed project will investigate the spatial and temporal regulatory molecular mechanisms of compartmentalized TNF-R1 signal transduction. We and others recently showed that TNF-triggered receptor internalization is an early event that regulates proliferative and apoptotic signal transduction. Recently, we found that internalization and intracellular trafficking as well as selective adaptor protein recruitment is dependent on the K63-ubiquitination of TNF-R1 by the E3 ubiquitin ligase RNF8. RNF8 knock-down resulted in enhanced proliferative and suppressed apoptotic signalling. In this grant proposal we will continue our research focusing on the following topics: (I.) How is TNF-R1 ubiquitination regulated: Is the recruitment of RNF8 preceded by receptor phosphorylation? Does palmitoylation of TNF-R1 play a role in the ubiquitination of the receptor? Which enzymes interact with RNF8 for TNF-R1 ubiquitination? Which are the phosphorylation-, palmitoylation- and ubiquitination-sites of endogenous TNF-R1? (II.) We will identify and characterize K63-ubiquitin binding proteins involved in TNF-receptor endocytosis and trafficking based on a proteomics screen of isolated early and late TNF-receptosomes. (III.) We want to characterize the molecular regulation of intracellular fusion of TNF-R1 receptosomes with trans-Golgi vesicles and the formation of multivesicular bodies containing TNF-receptosomes. (IV.) TNF-induced changes in the protein composition in other subcellular compartments (e.g. nuclei, cytosol, mitochondria and lysosomes) at different time points after TNF-treatment will be determined by 2D-differential gel electrophoresis (DIGE), followed by MS analysis. (V.) To get insight into the TNF-induced functional interactions of the proteins identified to be involved in TNF signaling from receptosomes, we will apply 2D blue-native gel-electrophoresis, western blot and mass spectrometric analysis. (VI.) The biological role of the identified novel components of the intracellular TNF-signalling will be determined in tumor cell lines that differ in their sensitivity to TNF-induced apoptosis. We will analyze TNF-R1 internalization and intracellular distribution of TNF-R1 receptosomes. We then want to define the expression of candidate proteins identified before and which could be responsible for the TNF resistance by western blotting. (VII.) In long term perspective, we plan to extend our analyses of the spatio-temporal regulation of compartmentalized signal transduction to other members of the TNF-receptor superfamily, CD95 and TRAIL-R1/R2, in order to define similarities and/or functional differences.From the envisioned experiments we expect a more detailed insight in the molecular mechanisms of proliferation and apoptosis induction via death receptors. In future this should add new informations for a targeted intervention with these signalling cascades in pathophysiological conditions such as cancer and inflammation.

• DFG Programme: Research Grants