Walczak Henning, Haas Tobias L
Division of Apoptosis Regulation, German Cancer Research Center, Im Neuenheimer Feld, Heidelberg, Germany.
Methods Mol Biol. 2008;414:221-39. doi: 10.1007/978-1-59745-339-4_16.
The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the proapoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway.
当肿瘤坏死因子（TNF）受体超家族（TNFRSF）的某些成员被其同源配体（属于TNF超家族（TNFSF））寡聚化时，外源性凋亡途径被激活。TNFRSF成员的凋亡诱导能力依赖于受体蛋白胞内部分存在死亡结构域（DD）。这类受体也被称为死亡受体。TNFSF配体与细胞表面表达的TNFRSF受体结合会导致受体近端蛋白复合物的形成。这种蛋白复合物是细胞内进一步信号转导事件的平台。对于像TNF相关凋亡诱导配体受体1（TRAIL-R1/DR4）、TRAIL-R2（KILLER/APO-2/DR5/TRICK）、CD95（Fas，APO-1）或TNF受体1（TNF-R1）这样的死亡受体，这种复合物被称为死亡诱导信号复合物（DISC）。在过去12年中，对各种DISC的组成进行了深入研究。对于CD95和TRAIL-R1/R2 DISC，现在已经清楚衔接蛋白Fas相关DD蛋白（FADD）是这些复合物的一部分，并且对于募集促凋亡信号分子半胱天冬酶-8和半胱天冬酶-10是必需的。这些蛋白酶的募集使其在DISC处被激活并随后诱导凋亡。半胱天冬酶-8同源的细胞FLICE样抑制蛋白（cFLIP）也可被募集到DISC。cFLIP通过干扰DISC处半胱天冬酶8和10的激活而作为一种抗凋亡调节因子发挥作用。有趣的是，用传统化疗药物或蛋白酶体抑制剂处理对TRAIL耐药的肿瘤细胞会使这些细胞对TRAIL诱导的凋亡敏感。通过应用此处描述的TRAIL DISC的生化分析方法，我们能够表明这种致敏主要是由于作为外源性途径凋亡起始蛋白复合物的DISC的生化组成变化。
