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 （TNFR1）和TNFR2起作用。对TNFR1的研究导致对TNF信号传导的许多方面的详细了解，并导致对细胞死亡和炎症研究的普遍相关性的发现。然而，TNFR2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TNFR2信号传导的理解，从而更深入地了解TNF生物学。在此背景下，我们在计划已届满的资助期内作出以下主要调查结果：首先，我们确定TNFR2是MAP3激酶NIK和NFkappaB途径的激活剂。此外，我们确定了去泛素酶Cyld和Sharpin（线性泛素链组装复合物的一个亚基）作为TNFR2信号复合物的新组分。我们还首次证明了TNFR2调节非凋亡TNFR1信号，并揭示了TNFR2和TWEAK受体Fn14 （TNF受体家族的另一成员）之间相当大的功能相似性。根据这些结果，在第二个资助期应解决以下问题：1。NIK和IKK1的激活是激活可选NFkappaB通路的关键步骤，但对于这两种分子，特别是在Notch和Wnt信号通路中，NFkappaB独立的功能也已被描述。基于我们的发现，TNFR2激活NIK以及可选的NFkappaB通路，我们将评估TNFR2与Notch和/或Wnt信号之间是否存在串扰。我们确定Cyld和Sharpin是TNFR2信号复合体的一部分，但还没有关于它们在TNFR2信号转导中的作用的信息。因此，我们将分析Cyld和Sharpin敲低在我们已建立的TNFR2信号传导细胞系统中的作用。如果我们能够在项目过程中确定tnfr2诱导的NIK-和ikk1介导的nfkappab -独立细胞效应，这些发现也将分析Cyld和Sharpin的可能作用。除了极少数例外，TNFR2信号复合体的所有成分都是通过TRAF2间接募集的，TRAF2是一种具有E3连接酶活性的多功能接头蛋白。为了破译TRAF2在TNFR2信号传导中的复杂功能，我们将生成TRAF2敲低/敲除细胞，并用已知的TRAF2突变体重建它们，这些突变体允许解剖TRAF2相关功能。TNFR2主要在免疫细胞和内皮细胞中表达。迄今为止，我们在TNFR2信号传导方面的工作得出的结果和想法主要基于肿瘤细胞系的使用。因此，我们将在人类和小鼠来源的相应原代细胞中重新评估这些发现。小鼠原代细胞的使用也将允许对Sharpin或NIK突变小鼠的细胞进行分析。

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.