Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.

中文翻译：

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Gasdermin D 半胱氨酸残基协同控制其棕榈酰化介导的膜靶向和组装。


Gasdermin D （GSDMD） 通过组装成透化质膜的寡聚体来执行细胞焦亡程序。在这里，通过单分子成像，我们阐明了 Gasdermin D 孔组装的尚不清楚的机制以及半胱氨酸残基在 GSDMD 寡聚化中的作用。我们表明 GSDMD 在膜上预组装成二聚体和三聚体结构单元，这些结构单元可以插入膜中，也可以在插入膜之前进一步组装成更高阶的寡聚体。GSDMD 残基 Cys39、Cys57 和 Cys192 是唯一参与 GSDMD 寡聚化的相关半胱氨酸。Cys192 的 S-棕榈酰化，结合带负电荷的脂质的存在，控制 GSDMD 膜靶向。同时发生的 Cys39/57/192 到丙氨酸 （Ala） 突变，而不是 Cys192 或 Cys39/57 对的 Ala 突变，完全消除了 GSDMD 插入人工膜和质膜。最后，Cys192 或 Cys39/Cys57 对足以形成 GSDMD 二聚体/三聚体，但它们都是功能性高阶寡聚体形成所必需的。总体而言，我们的研究揭示了 Cys192 棕榈酰化介导的膜结合和 Cys39/57/192 介导的寡聚化在 GSDMD 孔组装中的协同作用。本研究支持一种模型，其中 Gasdermin D 寡聚化依赖于由特定半胱氨酸残基介导的两步机制。