Two Adjacent Trimeric Fas Ligands Are Required for Fas Signaling and Formation of a Death-Inducing Signaling Complex

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.膜结合型Fas配体（Fas ligand, FasL）可通过结合死亡受体Fas，向靶细胞传递凋亡信号；而经蛋白水解加工得到的可溶性FasL则无法诱导细胞死亡。不过，可溶性FasL经交联后可被激活。由于具有细胞毒性的FasL寡聚化最低程度尚不明确，我们工程化构建了在同一分子内包含两个FasL三聚体的六聚体蛋白，具体通过将FasL分别与免疫球蛋白G1（immunoglobulin G1, IgG1）的Fc段，或ACRP30/脂联素（adiponectin）的胶原结构域融合实现。三聚体FasL与六聚体FasL均可结合Fas，但仅六聚体形式展现出高细胞毒性，且能够通过形成死亡诱导信号复合物（death-inducing signaling complex, DISC）介导凋亡信号传递。我们得以解析Fas介导的细胞凋亡过程中的三个连续早期事件，即受体结合、受体激活与胞内信号分子募集，且每一步均独立于后续步骤发生。本研究结果表明，FasL的有限寡聚化——极大概率也适用于CD40L等其他肿瘤坏死因子家族配体——是触发其信号通路的必要条件。