The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

细胞焦亡过程由炎症小体和下游效应子gasdermin D (GSDMD) 介导。被炎症小体相关的半胱天冬酶裂解后，GSDMD 的 N 末端结构域形成促进细胞溶解的膜孔。许多蛋白质促进 GSDMD 裂解，但已知没有一种蛋白质是 GSDMD 裂解后孔形成所必需的。在此，我们报告了一项正向遗传筛选，确定 Ragulator-Rag 复合物对于巨噬细胞中 GSDMD 孔形成和焦亡是必需的。机制分析表明，Ragulator-Rag 不是炎症小体激活时 GSDMD 裂解所必需的，而是促进质膜中 GSDMD 寡聚化。 GSDMD 寡聚化和孔形成中的缺陷可以通过刺激活性氧 (ROS) 产生的线粒体毒物来修复，并且 ROS 调节会影响炎性体途径促进 GSDMD 裂解下游孔形成的能力。这些发现揭示了免疫的关键调节因子（炎性体-GSDMD）和代谢（Ragulator-Rag）之间存在意想不到的联系。