Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, Korea.
Metabolic Regulation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, Korea.
Oncogene. 2018 Sep;37(36):4994-5006. doi: 10.1038/s41388-018-0323-z. Epub 2018 May 23.
Fas-associated death domain (FADD) is an adaptor protein recruiting complexes of caspase 8 to death ligand receptors to induce extrinsic apoptotic cell death in response to a TNF superfamily member. Although, formation of the complex of FADD and caspase 8 upon death stimuli has been studied in detail, posttranslational modifications fine-tuning these processes have yet to be identified. Here we revealed that K6-linked polyubiquitylation of FADD on lysines 149 and 153 mediated by C terminus HSC70-interacting protein (CHIP) plays an important role in preventing formation of the death inducing signaling complex (DISC), thus leading to the suppression of cell death. Cells depleted of CHIP showed higher sensitivity toward death ligands such as FasL and TRAIL, leading to upregulation of DISC formation composed of a death receptor, FADD, and caspase 8. CHIP was able to bind to FADD, induce K6-linked polyubiquitylation of FADD, and suppress DISC formation. By mass spectrometry, lysines 149 and 153 of FADD were found to be responsible for CHIP-mediated FADD ubiquitylation. FADD mutated at these sites was capable of more potent cell death induction as compared with the wild type and was no longer suppressed by CHIP. On the other hand, CHIP deficient in E3 ligase activity was not capable of suppressing FADD function and of FADD ubiquitylation. CHIP depletion in ME-180 cells induced significant sensitization of these cells toward TRAIL in xenograft analyses. These results imply that K6-linked ubiquitylation of FADD by CHIP is a crucial checkpoint in cytokine-dependent extrinsic apoptosis.
Fas 相关死亡结构域（FADD）是一种衔接蛋白，它可募集半胱天冬酶 8 复合物至死亡配体受体，以响应肿瘤坏死因子超家族成员诱导外在凋亡细胞死亡。尽管在死亡刺激下 FADD 和半胱天冬酶 8 复合物的形成已被详细研究，但微调这些过程的翻译后修饰尚未被鉴定。在这里，我们揭示了 C 端热休克蛋白 70 相互作用蛋白（CHIP）介导的 FADD 赖氨酸 149 和 153 上的 K6 连接多泛素化在防止死亡诱导信号复合物（DISC）形成中起着重要作用，从而导致细胞死亡受到抑制。耗尽 CHIP 的细胞对 FasL 和 TRAIL 等死亡配体表现出更高的敏感性，导致由死亡受体、FADD 和半胱天冬酶 8 组成的 DISC 形成上调。CHIP 能够与 FADD 结合，诱导 FADD 的 K6 连接多泛素化，并抑制 DISC 的形成。通过质谱分析，发现 FADD 的赖氨酸 149 和 153 负责 CHIP 介导的 FADD 泛素化。与野生型相比，这些位点发生突变的 FADD 能够更有效地诱导细胞死亡，并且不再受 CHIP 抑制。另一方面，缺乏 E3 连接酶活性的 CHIP 不能抑制 FADD 功能和 FADD 泛素化。在 ME-180 细胞中耗尽 CHIP 会导致这些细胞在异种移植分析中对 TRAIL 的敏感性显著增加。这些结果表明，CHIP 对 FADD 的 K6 连接泛素化是细胞因子依赖性外在凋亡的关键检查点。
