Control of gasdermin D oligomerization and pyroptosis by the Ragulator-Rag-mTORC1 pathway. Mus musculus breed:R26-Cas9 Knock In

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（pyroptosis）的过程由炎性体（inflammasomes）及下游效应蛋白焦亡蛋白D（gasdermin D, GSDMD）介导。当被炎性体相关的半胱天冬酶（caspases）切割后，GSDMD的N端结构域会形成膜孔，进而促进细胞裂解。目前已发现多种蛋白可促进GSDMD的切割，但尚未有蛋白被证实对GSDMD切割后的孔道形成是必需的。本研究通过正向遗传筛选（forward genetic screen），鉴定出Ragulator-Rag复合物（Ragulator-Rag complex）是巨噬细胞中GSDMD孔道形成与细胞焦亡所必需的调控因子。机制分析显示，Ragulator-Rag复合物并非炎性体激活时GSDMD切割所必需，而是可促进GSDMD在质膜上发生寡聚化。GSDMD寡聚化与孔道形成的缺陷，可通过刺激活性氧（reactive oxygen species, ROS）生成的线粒体毒物进行挽救，且活性氧的调控会影响炎性体通路在GSDMD切割下游促进孔道形成的能力。本研究揭示了免疫关键调控通路（炎性体-GSDMD）与代谢通路（Ragulator-Rag）之间此前未被发现的关联。