Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

混合谱系激酶结构域样 (MLK​​L) 是坏死性凋亡的重要分子，坏死性凋亡是一种由质膜直接破坏引发的细胞死亡过程。在坏死性凋亡期间，MLKL 被受体相互作用蛋白激酶 3（RIPK3 或 RIP3）磷酸化，然后转移到质膜以破坏膜的完整性。最近的数据表明，MLKL 在管腔内和细胞外囊泡 (EV) 的生成中以及在促进坐骨神经再生时的髓鞘破坏中也具有 RIP3 固有功能。在这里，我们展示了 MLKL 的消耗以不依赖 RIP3 的方式增强了 TRAIL 诱导的细胞死亡。MLKL 的消耗导致细胞毒性信号延长，从而增加 TRAIL 诱导的细胞死亡。最初，TRAIL 与细胞表面的 DR5 结合，并在表达 MLKL 和耗尽 MLKL 的细胞中以相似的速率被内吞，溶酶体对细胞内 TRAIL 的最终降解在 MLKL 耗尽的细胞中被延迟，对应于延长/增强的细胞内信号，例如 p -ERK 和 p-p38 在这些细胞中。TRAIL 与早期内体标志物 EEA1 的共定位表明，与表达 MLKL 的细胞相比，TRAIL 在 MLKL 耗尽细胞的早期内体中积累。这表明 MLKL 的消耗减少了受体-配体内体运输，导致 TRAIL-细胞毒性增加。一个 MLKL 突变体损害了其坏死性功能和其在 EV 产生中的功能，足以挽救 MLKL 缺乏症，表明这些功能所需的 N 端结构元件不是 MLKL 在与调节死亡受体细胞毒性相关的细胞内运输中的功能所必需的。因此，预计癌细胞中 MLKL 表达的减少会导致增强的 TRAIL 诱导的治疗功效。