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

在本项目中，我们旨在利用我们的专利方法通过免疫磁分选分离完整的亚细胞膜区室，评估死亡受体TNF-R1， CD95， TRAIL受体1和-2激活酸性鞘磷脂酶（A-SMase）的分子机制。首先，我们将分析反式高尔基囊泡与内化的tnf受体融合导致A-SMase在内体/多囊室晚期活化的机制。接下来，我们想要研究TNF- r1相关核黄素激酶在TNF受体体中介导的活性氧（ROS）对A-SMase激活的贡献。在第二部分中，我们将鉴定CD95和TRAIL响应的含A-SMase的小室，鉴定这些小泡易位的启动信号，研究携带A-SMase的分泌小泡向质膜运输的调控，以及胞外分泌过程中/细胞表面暴露后A-SMase激活的机制。在这里，我们将重点关注A-SMase的转录后修饰。在第三部分，我们想要跟踪CD95-和trail -受体体的内化和运输，并要求激活内切溶酶体A-SMase和神经酰胺下游可能的细胞内效应分子。最后，我们的目的是研究神经酰胺代谢的区隔化和调控，包括鞘氨醇和鞘氨醇-1-磷酸在分离的TNF-R1、CD95和TRAIL受体体中的产生。

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.