GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes
GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes
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DOI：
10.1073/pnas.1607769113
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发表时间：
2016-07-12
影响因子：
11.1
通讯作者：
Dueber, Erin C.
中科院分区：
文献类型：
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作者：
Aglietti, Robin A.;Estevez, Alberto;Dueber, Erin C.
Gasdermin-D（GsdmD）是先天免疫防御的关键介质，因为其被炎性半胱天冬酶1、4、5和11切割产生诱导焦亡的N-末端p30片段，焦亡是一种对消除细胞内细菌重要的死亡程序。GsdmD p30如何确切地触发焦亡尚未确定。在这里，我们表明，人GsdmD p30膜内形成功能孔。当在脂质体存在下从相应的C-末端GsdmD p20片段中释放时，GsdmD p30定位于脂质双层，而p20保留在水性环境中。在脂质体中，p30作为高阶低聚物存在，并形成通过负染色电子显微镜观察到的环状结构。这些结构出现在GsdmD裂解的几分钟内，并从预载脂质体中释放出Ca 2+。与GsdmD p30有利于与膜结合一致，p30仅在脂多糖激活caspase-11后的永生化巨噬细胞的含膜部分中检测到。我们发现，小鼠I105 N/人I104 N突变，这已被证明可以防止巨噬细胞pyroptosis，衰减细胞杀伤p30在293 T瞬时过表达系统和膜透化在体外，这表明突变体实际上是hypomorphs，但必须高于一定的浓度表现出的活动。总的来说，我们的数据表明GsdmD p30通过形成损害细胞膜完整性的孔来杀死细胞。
Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within inutes of GsdmD cleavage and released Ca2+ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.