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

该项目将研究分区化的肿瘤坏死因子-1信号转导的空间和时间调控分子机制。我们和其他人最近发现，肿瘤坏死因子触发的受体内化是调节增殖和凋亡信号转导的早期事件。最近，我们发现细胞内化和细胞内转运以及选择性接头蛋白募集依赖于E3泛素连接酶RNF8对肿瘤坏死因子-R1的K63泛素化。RNF8基因敲除后，细胞增殖信号增强，细胞凋亡信号受抑制。在这份赠款提案中，我们将继续围绕以下主题进行研究：(一)肿瘤坏死因子-R1泛素化是如何调节的：受体磷酸化是否先于RNF8的募集？肿瘤坏死因子-R1的棕榈酰化是否在受体的泛素化过程中起作用？哪些酶与RNF8相互作用，使肿瘤坏死因子-R1泛素化？内源性肿瘤坏死因子-R1的磷酸化、棕榈酸化和泛素化是什么部位？(Ii)我们将基于分离的早期和晚期肿瘤坏死因子受体的蛋白质组学筛选，鉴定和表征参与肿瘤坏死因子受体内吞和转运的K63-泛素结合蛋白。(三)我们想要研究细胞内的肿瘤坏死因子受体1受体小体与跨高尔基体小泡融合的分子调控以及含有肿瘤坏死因子受体小体的多囊泡体的形成。(四)用双向差示凝胶电泳法(DGE)检测肿瘤坏死因子处理后不同时间点其他亚细胞亚室(如细胞核、胞浆、线粒体和溶酶体)中蛋白质组成的变化，然后进行MS分析。(五)为了深入了解受肿瘤坏死因子诱导的蛋白质之间的功能相互作用，我们将应用二维蓝天然凝胶电泳法、蛋白质印迹和质谱仪进行分析。(Vi)已识别的细胞内肿瘤坏死因子信号的新成分的生物学作用将在肿瘤细胞系中确定，这些肿瘤细胞系对肿瘤坏死因子诱导的凋亡的敏感性不同。我们将分析肿瘤坏死因子受体1的内化和在细胞内的分布。然后，我们希望通过Western blotting确定之前确定的候选蛋白的表达，这些候选蛋白可能与肿瘤坏死因子耐药有关。(Vii)从长远来看，我们计划将我们对区区化信号转导的时空调控的分析扩展到肿瘤坏死因子受体超家族的其他成员，CD95和TRAIL-R1/R2，以确定相似之处和/或功能差异。从设想的实验中，我们期待着对死亡受体诱导细胞增殖和凋亡的分子机制有更详细的了解。在未来，这将为对癌症和炎症等病理生理条件下的这些信号级联进行有针对性的干预增加新的信息。

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.