Activation of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) by heteromeric ligands of the TNF superfam,ily (TNFSF)

TNF超家族（TNFSF）的配体，除LTa外，组装成同源三聚体，并作为II型跨膜和可溶性分子存在。除了充分研究的同源三聚体对称TNFSF配体之外，还存在一些不对称异源三聚体TNFSF配体。例如，LTa与LTa形成异源三聚体，并且我们在自己未发表的先前工作中发现了EDA-A1和EDA-A2之间的异源三聚体的形成。 同源三聚体配体对TNF受体超家族（TNFRSF）受体的激活分两步进行。首先，配体三聚体与三个受体分子相互作用。由此产生的配体-受体复合物不能强烈地触发细胞凋亡和经典的NF-κ B信号传导。事实上，后者需要最初形成的三聚体配体-受体复合物的次级相互作用。膜结合的同源三聚体TNFSF配体定期触发这第二步。然而，在可溶性TNFSF配体的情况下，配体-受体复合物的二次聚集取决于所考虑的受体类型。大多数可溶性TNFSF配体诱导的受体三聚体不能自发聚集，因此不能触发完全的受体活化。然而，少数具有强的内在自聚集能力的TNFRSF受体（例如TNFR 1）响应于可溶性配体分子而二次聚集，并允许完全的受体活化。异源三聚体TNFSF配体不能募集三个相同的TNFRSF受体分子，而是可以潜在地与两种类型的TNFRSF受体相互作用。如果仅表达一种受体类型，则异聚配体仅结合一种或两种受体分子。此外，还不清楚异源三聚体TNFSF配体是否充当其相应同源三聚体对应物的激动剂、修饰剂或拮抗剂。在TNFRSF受体活化的2步模型的背景下，这提出了异源三聚体配体分子如何在分子和细胞水平上起作用的问题。因此，我们将以LTa 2 β b、EDA-A1（EDA-A2）2和（EDA-A1）2 EDA-A2为例研究异三聚体TNFSF配体的作用模式。EDA-A1和EDA-A2在TNFSF中是独特的，除了特征性TNF同源结构域之外，还具有允许形成同源和异源EDA-A1/2六聚体的胶原结构域。因此，我们还将分析限定化学计量的六聚体EDA-A1/2变体的活性。 计划的研究不仅对了解LTa 2，EDA-A1和EDA-A2及其受体的生物学很重要，而且还可以提供异聚TNFSF分子功能的一般见解。后者可以帮助以合理的方式开发具有新的、潜在的治疗上有价值的性质的非天然存在的TNFSF配体变体，例如仅结合一种或两种受体原聚体的配体三聚体或TNFRSF受体-双特异性六聚体。

The ligands of the TNF superfamily (TNFSF), with exception of LTa, assemble into homotrimers and occur as type II transmembrane and as soluble molecules. Besides the well investigated homotrimeric symmetric TNFSF ligands, there are also a few asymmetric heterotrimeric TNFSF ligands. For example, LTa forms heteromers with LTß and we found in own unpublished previous work formation of heterotrimers between EDA-A1 and EDA-A2. The activation of receptors of the TNF receptor superfamily (TNFRSF) by homotrimeric ligands takes place in two steps. First, a ligand trimer interacts with three receptor molecules. The resulting ligand-receptor complexes are unable to robustly trigger apoptosis and classical NF-kappaB signaling. In fact, the latter needs secondary interaction of the initially formed trimeric ligand-receptor complexes. Membrane-bound homotrimeric TNFSF ligands regularly trigger this second step. In the case of soluble TNFSF ligands, however, secondary aggregation of ligand-receptor complexes is dependent on the receptor type considered. Most soluble TNFSF ligand-induced receptor trimers do not cluster spontaneously and thus fail to trigger full receptor activation. A few TNFRSF receptors with a strong intrinsic capacity to autoaggregate (e.g.TNFR1), however, secondarily aggregate in response to soluble ligand molecules and allow full receptor activation. Heterotrimeric TNFSF ligands are not able to recruit three identical TNFRSF receptor molecules but instead can potentially interact with two types of TNFRSF receptors. If only one receptor type is expressed, the heteromeric ligands only bind one or two receptor molecules. It is furthermore unclear whether heterotrimeric TNFSF ligands act as agonists, modifiers or antagonists of their corresponding homotrimeric counterparts. Against the background of the 2-step model of TNFRSF receptor activation, this raises the question how heterotrimeric ligand molecules act at the molecular and cellular level. We will therefore investigate the mode of action of heterotrimeric TNFSF ligands on the example of LTa2ßb, EDA-A1(EDA-A2)2 and (EDA-A1)2EDA-A2. EDA-A1 and EDA-A2 are unique in the TNFSF by having besides the characteristic TNF homology domain also a collagen domain which allows the formation of homo- and heteromeric EDA-A1/2 hexamers. We will therefore also analyze the activity of hexameric EDA-A1/2 variants of defined stoichiometry. The planed studies are not only important to understand the biology of LTa2, EDA-A1 and EDA-A2 and their receptors but could also provide general insights in the function of heteromeric TNFSF molecules. The latter could help to develop in a rational manner non-naturally occurring TNFSF ligand variants with novel, potentially therapeutically valuable properties, e.g. ligand trimers that bind only one or two receptor protomers or TNFRSF receptor-bispecific hexamers.