Mechanisms and principles of tumor necrosis factor (TNF)-receptor activation

肿瘤坏死因子（TNF）受体被TNF家族的配体激活。后者通常表现为三聚体膜结合分子，但也经常以可溶性形式出现，例如由于蛋白水解处理。结合膜结合TNF配体总是导致强TNF受体活化。然而，TNF受体对可溶性配体的反应不同。一些TNF受体类型通过结合可溶性配体分子（如TNFR1）而被强烈激活，而另一些受体类型结合可溶性配体但不能触发细胞内信号传导（如TNFR2）。一些TNF受体类型对可溶性配体缺乏反应性反映了受体的局限性，而不是配体分子的缺失质量。例如，可溶性TNF强烈触发TNFR1信号，但实际上对TNFR2无活性，尽管结合良好。我们之前工作的一个基本观察是，最初形成的三聚体TNF配体受体复合物的二次聚集代表了激活各种TNF受体相关途径的重要步骤，即使不是必要步骤。通过各种实验数据，这一概念支持那些在可溶性配体三聚体结合后不被激活或仅被激活不良的TNF受体类型。然而，被可溶性配体很好激活的TNF受体类型是否也形成并利用超分子配体受体簇进行信号转导尚不清楚。以TNFR1为例，本项目将通过双分子发光互补实验、共免疫沉淀实验和单分子跟踪荧光显微镜来解决这一基本问题。我们在未发表的工作中发现，TNFR1的单体可溶性外结构域，即受体的脱落形式，已经与TNF高亲和力相互作用。因此，我们将澄清这些可溶性受体分子是否被纳入三聚体和低聚体TNF配体受体复合物中，并以显性负性方式在那里起作用。基于自己的数据和来自文献的初步证据，我们将进一步评估CD95和TNFR2不同的TNF受体相关信号通路被三聚体和寡聚体配体受体复合物不同激活的可能性。为此，我们将通过质谱法和western blotting技术对比分析TNFR2和CD95三聚体和低聚体信号复合物相关的信号蛋白的身份、比例和修饰。获得的研究结果将通过已建立的生化方法评估其与TNFR2和CD95信号的功能相关性。

Tumor necrosis factor (TNF) receptors become activated by ligands of the TNF family. The latter are typically expressed as trimeric membrane-bound molecules but often occur also in a soluble form, e.g. due to proteolytic processing. Binding of membrane-bound TNF ligands invariably results in strong TNF receptor activation. TNF receptors, however, differently respond to soluble ligands. Some TNF receptor types are strongly activated by binding of soluble ligand molecules (e.g. TNFR1), while others bind soluble ligands but fail to trigger intracellular signaling (e.g. TNFR2). The lack of responsiveness of some TNF receptor types towards soluble ligands mirrors a limitation of the receptor and not a missing quality of the ligand molecule. For example, soluble TNF strongly triggers TNFR1 signaling but is practical inactive on TNFR2 despite good binding.A basal observation in our previous work was that secondary aggregation of initially formed trimeric TNF ligand receptor complexes represents an important if not even obligate step in the activation of various TNF receptor-associated pathways. This concept is supported for those types of TNF receptors that become not or only poorly activated upon binding of soluble ligand trimers by various experimental data. However, it is unclear whether TNF receptor types that become quite well activated by soluble ligands also form and utilize supramolecular ligand receptor clusters for signal transduction. Using the example of TNFR1, this basic aspect will be addressed in the project by a bimolecular luminescence complementation assay, coimmunoprecipitation experiments and single-molecule tracking fluorescence microscopy.We found in unpublished work that the monomeric soluble ectodomain of TNFR1, thus the shedded form of the receptor, already interacts with high affinity with TNF. We will thus clarify whether such soluble receptor molecules are incorporated into trimeric and oligomeric TNF ligand receptor complexes and act there in a dominant negative fashion.Based on own data and initial evidence from the literature, we will further evaluate for CD95 and TNFR2 the possibility that distinct TNF receptor-associated signaling pathways are differentially activated by trimeric and oligomeric ligand receptor complexes. For this purpose, we will comparatively analyze the identity, proportion and modifications of signaling proteins associated with trimeric and oligomeric TNFR2 and CD95 signaling complexes by mass spectrometry and western blotting. The findings obtained will then be evaluated with respect to their functional relevance for TNFR2 and CD95 signaling by established biochemical approaches.