Identification and functional analysis of TNFR2-induced signaling complexes

TNFR2诱导的信号复合物的鉴定和功能分析

• 批准号: 58713751
• 负责人: Professor Dr. Harald Günther Wajant
• 金额: --
• 依托单位: Abteilung für Molekulare Innere Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/58713751?language=en
• 关键词: Identification; functional; analysis; TNFR2; induced

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.

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）发挥作用。对 TNFR1 的研究使人们对 TNF 信号传导的许多方面有了详细的了解，并导致与细胞死亡和炎症研究具有普遍相关性的发现。然而，人们对TNFR2信号转导的机制仍知之甚少。因此，该项目的主要目标是提高对 TNFR2 信号传导的理解，以更深入地了解 TNF 生物学。在此背景下，我们在该项目资助期限届满后得出以下主要发现。首先，我们确定 TNFR2 是 MAP3 激酶 NIK 和替代 NFkappaB 通路的激活剂。此外，我们还鉴定出去泛素酶 Cyld 和 Sharpin（线性泛素链组装复合物的一个亚基）作为 TNFR2 信号复合物的新成分。我们还首次证明 TNFR2 调节非凋亡 TNFR1 信号传导，并揭示了 TNFR2 与 TWEAK 受体 Fn14（TNF 受体家族的另一个成员）之间相当大的功能相似性。基于这些结果，应在第二个资助期解决以下问题：1。 NIK 和 IKK1 的激活是激活替代 NFkappaB 途径的关键步骤，但这两种分子的 NFkappaB 独立功能，特别是在 Notch 和 Wnt 信号传导途径中，也已被描述。基于我们发现TNFR2 激活NIK 以及替代的NFkappaB 通路，我们将评估TNFR2 与Notch 和/或Wnt 信号传导之间是否存在串扰。2．我们确定 Cyld 和 Sharpin 是 TNFR2 信号转导复合物的一部分，但尚未了解它们在 TNFR2 信号转导中的作用。因此，我们将分析 Cyld 和 Sharpin 敲低对我们已建立的 TNFR2 信号传导细胞系统的影响。如果我们能够在项目过程中识别 TNFR2 诱导的 NIK 和 IKK1 介导的 NFkappaB 独立细胞效应，那么这些发现也将针对 Cyld 和 Sharpin 的可能作用进行分析。 3. 除了极少数例外，TNFR2 信号复合物的所有成分都是通过 TRAF2（一种具有 E3 连接酶活性的多功能接头蛋白）间接招募的。为了破译 TRAF2 在 TNFR2 信号转导中的复杂功能，我们将生成 TRAF2 敲低/敲除细胞，并用已知的充分表征的 TRAF2 突变体重建它们，从而可以剖析 TRAF2 相关功能。 4． 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，我们将重新评估人类和小鼠来源的相应原代细胞中的这些发现。使用原代小鼠细胞还可以分析来自带有突变 Sharpin 或 NIK 的小鼠的细胞。