Identification and functional analysis of TNFR2-induced signaling complexes

肿瘤坏死因子（TNF）是一种多效性细胞因子，通过刺激两种功能不同的受体，即TNF受体-1（TNFR 1）和TNFR 2起作用。对TNFR 1的研究导致了对TNF信号传导的许多方面的详细了解，并导致了与细胞死亡和炎症研究相关的发现。然而，TNFR 2信号转导的机制仍然知之甚少。因此，该项目的主要目的是提高对TNFR 2信号传导的理解，以更深入地了解TNF生物学。在此背景下，我们在项目的资助期届满后得出以下主要结果。首先，我们确定TNFR 2作为MAP 3激酶NIK和替代NF κ B途径的激活剂。此外，我们确定了去泛素化酶Cyld和Sharpin，线性泛素链组装复合物的一个亚基，作为TNFR 2信号复合物的新组分。我们还首次证明了TNFR 2调节非凋亡TNFR 1信号传导，并揭示了TNFR 2和TNF受体家族的另一个成员TWEAK受体Fn 14之间相当大的功能相似性。NIK和IKK 1的激活是激活替代NF κ B途径的关键步骤，但对于这两种分子，也已经描述了NF κ B独立功能，特别是在Notch和Wnt信号传导途径中。基于我们发现TNFR 2激活NIK以及替代NF κ B通路，我们将评估TNFR 2与Notch和/或Wnt信号传导之间是否存在串扰。我们确定Cyld和Sharpin是TNFR 2信号复合物的一部分，但还没有关于它们在TNFR 2信号转导中作用的信息。因此，我们将分析Cyld和Sharpin敲低在我们建立的TNFR 2信号传导细胞系统中的作用。如果我们能够在项目过程中鉴定TNFR 2诱导的NIK和IKK 1介导的NF κ B非依赖性细胞效应，这些发现也将分析Cyld和Sharpin的可能作用。3.除了极少数例外，TNFR 2信号复合物的所有组分都是通过TRAF 2间接募集的，TRAF 2是一种具有E3连接酶活性的多功能衔接蛋白。为了破译TRAF 2在TNFR 2信号传导中的复杂功能，我们将产生TRAF 2敲低/敲除细胞，并用已知的充分表征的TRAF 2突变体重建它们，所述突变体允许剖析TRAF 2相关功能。TNFR 2主要在免疫细胞和内皮细胞中表达。迄今为止，我们对TNFR 2信号转导的研究结果和想法主要基于肿瘤细胞系的使用。因此，我们将在相应的人类和小鼠来源的原代细胞中重新评估这些发现。原代鼠细胞的使用还将允许分析衍生自具有突变的Sharpin或NIK的小鼠的细胞。

Tumor necrosis factor (TNF) is a pleiotropic cytokine acting through stimulation of two functional distinct receptors, namely TNF receptor-1 (TNFR1) and TNFR2. Studies on TNFR1 resulted in a detailed understanding of many aspects of TNF signaling and lead to discoveries of general relevance for research on cell death and inflammation. However, the mechanisms of TNFR2 signal transduction are still poorly understood. Therefore, it was the major aim of the project to improve the understanding of TNFR2 signaling to gain deeper insights into TNF biology in general. Against this background, we made the following major findings in the expired funding period of the project. First, we identified TNFR2 as an activator of the MAP3 kinase NIK and the alternative NFkappaB pathway. Further, we identified the deubiquitinase Cyld and Sharpin, a subunit of the linear ubiquitin chain assembly complex, as new components of the TNFR2 signaling complex. We also demonstrated for the first time that TNFR2 modulates non-apoptotic TNFR1 signaling and revealed considerable functional similarities between TNFR2 and the TWEAK receptor Fn14, another member of the TNF receptor family.Based on these results the following issues shall be addressed in the second funding period:1. Activation of NIK and IKK1 are pivotal steps in the activation of the alternative NFkappaB pathway but for both molecules NFkappaB-independent functions, particularly in the Notch and Wnt signaling pathways, have also been described. Based on our finding that TNFR2 activates NIK as well as the alternative NFkappaB pathway, we will evaluate whether there is a crosstalk between TNFR2 and Notch and/or Wnt signaling.2. We identified Cyld and Sharpin as parts of the TNFR2 signaling complex but have yet no information about their role in TNFR2 signal transduction. We will therefore analyze the effect of Cyld and Sharpin knockdown in our established cellular systems of TNFR2 signaling. If we should be able to identify TNFR2-induced NIK- and IKK1-mediated NFkappaB-independent cellular effects in the course of the project, these findings will also be analyzed with respect to a possible role of Cyld and Sharpin. 3. With very few exceptions, all components of the TNFR2 signaling complex are indirectly recruited via TRAF2, a multifunctional adaptor protein with E3 ligase activity. To decipher the complex functions of TRAF2 in TNFR2 signaling, we will generate TRAF2 knockdown/knockout cells and reconstitute them with known well characterized TRAF2 mutants that allow dissection of TRAF2-related functions.4. TNFR2 is primarily expressed in immune cells and endothelial cells. The results and ideas derived from our work on TNFR2 signaling mainly base so far on the use of tumor cell lines. We will therefore reevaluate these findings in corresponding primary cells of human and murine origin. The use of primary murine cells will also allow an analysis of cells derived from mice with mutated Sharpin or NIK.