Characterisation of FADD interactome reveals novel insights into FADD recruitment and signalling at the Death Inducing Signalling Complex (DISC)
Characterisation of FADD interactome reveals novel insights into FADD recruitment and signalling at the Death Inducing Signalling Complex (DISC)
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FADD 相互作用组的表征揭示了对 FADD 招募和死亡诱导信号复合体 (DISC) 信号传导的新见解
DOI：
10.1101/2021.03.25.436271
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Fox J
中科院分区：
文献类型：
--
作者：
Fox J
fas相关死亡结构域蛋白（FADD）在外源性凋亡通路中起着至关重要的作用，它是死亡诱导信号复合体（DISC）的重要组成部分。然而，促进FADD向DISC募集的精确的早期分子事件仍然不清楚。我们利用亲和纯化和质谱技术研究了未处理细胞和死亡受体刺激后的FADD相互作用蛋白，以鉴定新的FADD相互作用蛋白。正如预期的那样，在死亡受体刺激的样本中，我们的分析确定了DISC的关键成分，如Caspase-8。此外，我们发现了新的结合伙伴，包括转铁蛋白受体1 （TfR1）和肌球蛋白轻链激酶2 (MYLK2)，它们能够调节FADD向DISC的募集，从而调节下游凋亡信号传导。TfR1与FADD预先相关，并被招募到DISC中；此外，我们的数据显示，TfR1也与死亡受体TRAIL-R1和TRAIL-R2预相关，从而作为DISC形成的关键调节因子。在MYLK2的情况下，非凋亡细胞中FADD与MYLK2的特异性结合将FADD与其他DISC成分隔离，确保不启动异常凋亡。此外，MYLK2的酶活性需要使其与FADD一起转运到disc介导的死亡受体寡聚化位点。综上所述，我们的研究强调了新的FADD结合伙伴在调节死亡受体介导的凋亡细胞死亡中发挥的重要作用，部分是通过调节FADD向DISC的募集。
Fas-associated death domain protein (FADD) plays a vital role in the extrinsic apoptotic pathway, where it forms an essential component of the death-inducing signaling complex (DISC). However, the precise early molecular events that facilitate recruitment of FADD to the DISC remain poorly defined. Using affinity purification and mass spectrometry we investigated the FADD interactome in untreated cells and following death receptor stimulation to identify novel FADD-interacting proteins. As expected, in death receptor-stimulated samples our analysis identified key components of the DISC such as Caspase-8. In addition, we identified novel binding partners including Transferrin Receptor 1 (TfR1) and Myosin Light Chain Kinase 2 (MYLK2) that are able to modulate FADD recruitment to the DISC and consequently downstream apoptotic signaling. TfR1 is pre-associated with FADD and recruited into the DISC; moreover, our data reveal that TfR1 is also pre-associated with the death receptors, TRAIL-R1 and TRAIL-R2, thereby functioning as a key regulator of DISC formation. In the case of MYLK2, specific binding of FADD to MYLK2 in non-apoptotic cells sequesters FADD from other DISC components ensuring aberrant apoptosis is not initiated. Furthermore, MYLK2 enzymatic activity is required to for it to translocate, in complex with FADD, to sites of DISC-mediated death receptor oligimerization. Taken together, our study highlights the important role that additional novel FADD binding partners play in the regulation of death receptor-mediated apoptotic cell death, in part by modulating FADD recruitment to the DISC.
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