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，除了LTa外，组装成同型三聚体，并以II型跨膜和可溶性分子的形式出现。除了研究较多的同三聚体对称TNFSF配体外，还有一些不对称的异三聚体TNFSF配体。例如，LTa与LTß形成异聚体，我们在自己未发表的先前工作中发现EDA-A1和EDA-A2之间形成异聚三聚体。同源三聚体配体激活TNF受体超家族（TNFRSF）的受体分两个步骤进行。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-受体复合物不能强有力地触发细胞凋亡和经典的nf - κ b信号传导。实际上，后者需要初始形成的三聚体配体-受体复合物的二次相互作用。膜结合的三聚体TNFSF配体通常会触发第二步。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，因此不能触发受体的完全激活。少数TNFRSF受体具有很强的内在自聚集能力（如tnfr1），然而，在可溶性配体分子的响应下，二级聚集并允许受体完全激活。异三聚体TNFRSF配体不能招募三个相同的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。如果只表达一种受体类型，则异聚体只结合一种或两种受体分子。此外，还不清楚异三聚体tnf - sf配体是否作为其相应的同三聚体的激动剂、修饰剂或拮抗剂。在TNFRSF受体激活的两步模型的背景下，这提出了异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LTa2ßb、EDA-A1(EDA-A2)2和（EDA-A1）2EDA-A2为例研究异三聚体TNFSF配体的作用方式。EDA-A1和EDA-A2在TNF - sf中是独一无二的，除了具有典型的TNF同源结构域外，还有一个胶原结构域，该结构域允许形成同源和异聚的EDA-A1/2六聚体。因此，我们也将分析定义化学计量学的六聚体EDA-A1/2变体的活性。这些计划中的研究不仅对了解LTa2、EDA-A1和EDA-A2及其受体的生物学特性具有重要意义，而且还可以对异质TNFSF分子的功能提供一般的见解。后者可以帮助以合理的方式开发具有新颖、潜在治疗价值的非自然发生的TNFSF配体变体，例如配体三聚体仅结合一种或两种受体原体或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 For example, LTa forms heteromers with LTß and we found in own unpublished previous work formation of heterotrimers between EDA-A1 and EDA-A2.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.