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

肿瘤坏死因子（TNF）受体激活的机制和原理

• 批准号: 232775293
• 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: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232775293?language=en
• 关键词: Mechanisms; principles; tumor; necrosis; factor

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assay, coimmunoprecipitation experiments and single-molecule tracking fluorescence microscopy.We found in unpublished work that the monomeric soluble ectodomain of TNFR1, thus the shedded form of the receptor, already interacts with high affinity with TNF.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.

肿瘤坏死因子（TNF）受体被TNF家族的配体激活。后者通常表达为三聚体膜结合分子，但通常也以可溶形式存在，例如由于蛋白水解加工。膜结合的TNF配体的结合总是导致强烈的TNF受体活化。然而，TNF受体对可溶性配体的反应不同。一些TNF受体类型通过可溶性配体分子的结合而被强烈激活（例如TNFR1），而其他TNF受体类型结合可溶性配体但不能触发细胞内信号传导（例如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强烈地触发TNFR1信号传导，但尽管结合良好，但对TNFR2实际上是无活性的。我们先前工作中的一个基本观察是，最初形成的三聚体TNF配体受体复合物的二次聚集代表了多种TNF受体相关途径活化中的重要步骤，即使不是强制性步骤。通过各种实验数据，对于那些在可溶性配体三聚体结合后不被激活或仅被弱激活的TNF受体类型，这一概念得到支持。然而，目前还不清楚是否成为相当好的激活可溶性配体的TNF受体类型也形成和利用超分子配体受体簇的信号转导。以TNFR1为例，这个基本方面将在该项目中通过双分子发光互补测定、共免疫沉淀实验和单分子跟踪荧光microscopy.We发现在未发表的工作中，TNFR1的单体可溶性胞外域，因此受体的脱落形式，已经与TNF高亲和力相互作用。因此，我们将澄清这种可溶性受体分子是否被纳入三聚体和寡聚体TNF配体受体复合物，并在那里发挥作用，在一个显性负fashion.Based自己的数据和初步证据，从文献中，我们将进一步评估CD95和TNFR2的可能性，不同的TNF受体相关的信号转导途径被差异激活的三聚体和寡聚体配体受体复合物。为此，我们将比较分析的身份，比例和修改的信号蛋白与三聚体和寡聚体TNFR2和CD95信号复合物通过质谱和蛋白质印迹。然后将通过已建立的生物化学方法评价所获得的结果与TNFR2和CD95信号传导的功能相关性。