Topology, regulation and function of acid sphingomyelinase in death receptor signaling

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In this project we aim to evaluate the molecular mechanisms of acid sphingomyelinase (A-SMase) activation by the death receptors TNF-R1, CD95, TRAIL receptor 1 and -2 using our patented method to isolate intact subcellular membrane compartments by immunomagnetic sorting. First, the mechanism of the fusion of trans-Golgi vesicles with internalized TNF-receptosomes leading to A-SMase activation in late endosomal/multivesicular compartments will be analyzed. Next we want to investigate the contribution of reactive oxygen species (ROS) mediated by TNF-R1 associated riboflavin kinase within TNF receptosomes to A-SMase activation. In the second part, we will identify the CD95- and TRAIL- responsive A-SMase-containing compartment, identify the signal initiating translocation of these vesicles, study the regulation of trafficking of A-SMase-bearing secretory vesicles to the plasma-membrane, and the mechanisms of A-SMase activation during exocytosis / after exposure of the enzyme at the cell surface. Here we will focus on post-transcriptional modifications of A-SMase. In the third part, we want to follow internalization and trafficking of CD95- and TRAIL-receptosomes and ask for activation of endo-lysosomal A-SMase and possible intracellular effector molecules downstream of ceramide in this compartment. Finally, we aim to investigate the compartmentalization and regulation of ceramide metabolism including sphingosine and sphingosine-1-phospate production in isolated TNF-R1, CD95 and TRAIL receptosomes.