Necroptosis is a lytic and pro-inflammatory form of programmed cell death executed by the terminal effector, the MLKL (mixed lineage kinase domain-like) pseudokinase. Downstream of death and Toll-like receptor stimulation, MLKL is trafficked to the plasma membrane via the Golgi-, actin- and microtubule-machinery, where activated MLKL accumulates until a critical lytic threshold is exceeded and cell death ensues. Mechanistically, MLKL's lytic function relies on disengagement of the N-terminal membrane-permeabilising four-helix bundle domain from the central autoinhibitory brace helix: a process that can be experimentally mimicked by introducing the R30E MLKL mutation to induce stimulus-independent cell death. Here, we screened a library of 429 kinase inhibitors for their capacity to block R30E MLKL-mediated cell death, to identify co-effectors in the terminal steps of necroptotic signalling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJI308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits mammalian target of rapamycin (mTOR) signalling or regulators thereof, and blocked constitutive cell death executed by R30E MLKL. Our study implicates mTOR signalling as an auxiliary factor in promoting the transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death.

中文翻译：

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抑制剂确定了 mTOR 信号转导在 MLKL 激活下游坏死性凋亡执行中的辅助作用。


坏死性凋亡是一种溶解和促炎形式的程序性细胞死亡，由末端效应子 MLKL （混合谱系激酶结构域样） 假激酶执行。在死亡和 Toll 样受体刺激的下游，MLKL 通过高尔基体、肌动蛋白和微管机制运输到质膜，在那里激活的 MLKL 积累，直到超过临界裂解阈值并接踵而至细胞死亡。从机制上讲，MLKL 的裂解功能依赖于 N 末端膜透化四螺旋束结构域与中央自抑制支撑螺旋的脱离：这一过程可以通过引入 R30E MLKL 突变来诱导不依赖刺激的细胞死亡，从而在实验上模拟。在这里，我们筛选了一个包含 429 种激酶抑制剂的文库，以了解它们阻断 R30E MLKL 介导的细胞死亡的能力，以确定坏死性凋亡信号末端步骤中的协同效应器。我们鉴定了 13 种化合物 - ABT-578、AR-A014418、AZD1480、AZD5363、Idelalisib、Ipatasertib、LJI308、PHA-793887、雷帕霉素、利达福莫司、SMI-4a、替西罗莫司和泰德格鲁西 - 每一种化合物都抑制哺乳动物雷帕霉素靶标 （mTOR） 信号或其调节剂，并阻断 R30E MLKL 执行的组成型细胞死亡。我们的研究表明 mTOR 信号传导是促进活化的 MLKL 寡聚体转运到质膜的辅助因子，在那里它们积累成热点，使脂质双层透化，导致细胞死亡。