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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

肿瘤坏死因子受体 1 (TNF-R1) 内化和细胞内转运的调节及其对 TNF 信号转导和生物学结果的影响

基本信息

批准号：
269124904
负责人：
Professor Dr. Stefan Schütze
金额：
--
依托单位：
Institut für Immunologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2017-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/269124904?language=en
关键词：
Regulation internalization intracellular transport Tumor

项目摘要

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.

该项目将研究TNF-R1信号转导的空间和时间调控分子机制。我们和其他人最近表明，肿瘤坏死因子触发的受体内化是一个早期事件，调节增殖和凋亡信号转导。最近，我们发现，内化和细胞内运输以及选择性衔接蛋白的招聘是依赖于K63泛素化的TNF-R1的E3泛素连接酶RNF 8。RNF 8敲低导致增强的增殖和抑制的凋亡信号传导。在本研究计划中，我们将继续研究以下课题：（一）TNF-R1泛素化是如何调节的：RNF 8的募集是否先于受体磷酸化？TNF-R1的棕榈酰化在受体的泛素化中发挥作用吗？哪些酶与RNF 8相互作用以进行TNF-R1泛素化？内源性TNF-R1的磷酸化、棕榈酰化和泛素化位点是什么？（二）我们将确定和表征K63-泛素结合蛋白参与TNF受体的内吞作用和贩运的基础上分离的早期和晚期TNF受体体的蛋白质组学筛选。（三）我们希望表征TNF-R1受体体与高尔基体囊泡的细胞内融合的分子调节和含有TNF受体体的多泡体的形成。（四）在TNF处理后的不同时间点，TNF诱导的其他亚细胞区室（例如细胞核、胞质溶胶、线粒体和溶酶体）中蛋白质组成的变化将通过2D-差示凝胶电泳（DIGE）测定，然后进行MS分析。（五）为了深入了解TNF诱导的蛋白质的功能相互作用，确定参与TNF信号从受体体，我们将应用二维蓝色天然凝胶电泳，蛋白质印迹和质谱分析。（六）将在对TNF诱导的细胞凋亡的敏感性不同的肿瘤细胞系中确定所鉴定的细胞内TNF信号传导的新组分的生物学作用。我们将分析TNF-R1的内化和TNF-R1受体体的细胞内分布。然后，我们希望通过蛋白质印迹法来确定之前鉴定的候选蛋白质的表达，这些蛋白质可能是TNF抗性的原因。（七）从长远的角度来看，我们计划将我们的分析的时空调控区室化信号转导的TNF受体超家族的其他成员，CD 95和TRAIL-R1/R2，以确定的相似性和/或功能的差异，从设想的实验，我们期望在增殖和凋亡诱导通过死亡受体的分子机制更详细的洞察。在未来，这将为在癌症和炎症等病理生理条件下对这些信号级联进行靶向干预提供新的信息。