坏死性凋亡是由末端效应子执行的程序性细胞死亡的裂解和促炎形式,MLKL(混合谱系激酶结构域样)假激酶。死亡下游和Toll样受体刺激,MLKL通过Golgi被贩运到质膜-,动-和微管机械,其中激活的MLKL积累直到超过临界裂解阈值并且随后发生细胞死亡。机械上,MLKL的裂解功能依赖于N末端膜透化四螺旋束(4HB)结构域与中央自动抑制支撑螺旋的脱离:该过程可以通过引入R30EMLKL突变以诱导刺激-非依赖性细胞死亡进行实验模拟。这里,我们筛选了一个429个激酶抑制剂的文库,其阻断R30EMLKL介导的细胞死亡的能力,以识别坏死信号传导的终末步骤中的共效应子。我们鉴定了13个化合物-ABT-578,AR-A014418,AZD1480,AZD5363,Idelalisib,Ipatasertib,LJ1308,PHA-793887,雷帕霉素,Ridaforolimus,SMI-4a,坦西罗莫司和Tideglusib-各自抑制mTOR信号或其调节因子,并阻断R30EMLKL执行的组成型细胞死亡。我们的研究提示mTOR信号是促进激活的MLKL寡聚体向质膜转运的辅助因素。在那里它们积累到渗透脂质双层的热点中,导致细胞死亡。
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 (4HB) 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 signaling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJ1308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits 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 transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death.