Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Kennedy Institute of Rheumatology, NDORMS, University of Oxford, Oxford, UK.
Methods Mol Biol. 2022;2523:209-237. doi: 10.1007/978-1-0716-2449-4_14.
Cytosolic pattern recognition receptors trigger pyroptosis by detection of danger- or pathogen-associated molecular patterns. These receptors initiate the assembly of inflammasomes, multimeric protein complexes that drive caspase-1 activation. Active caspase-1 cleaves the proinflammatory cytokines IL-1β and IL-18 and the pore-forming protein gasdermin-D (GSDMD) thereby liberating its N-terminal domain. The GSDMD N-termini form multimeric pores at the plasma membrane that allow leakage of intracellular content and ultimately trigger a type of cell death called "pyroptosis." Emerging studies have revealed that GSDMD is also processed by apoptotic caspases-8/-3/-7. In this chapter, we aim to describe methods to monitor lytic cell death and to distinguish between GSDMD processing events and the GSDMD fragments that are generated after pyroptosis or apoptosis induction. We also illustrate the difference between GSDMD pore formation, and final cell lysis, and how this affects to the release of intracellular content. Finally, we show that the activation of another pore-forming protein, gasdermin-E, does not exclusively translate into lytic cell death in bone marrow-derived macrophages.
细胞质模式识别受体通过检测危险相关或病原体相关分子模式触发细胞焦亡。这些受体启动炎性小体的组装，炎性小体是一种多聚体蛋白复合物，可驱动半胱天冬酶-1 的激活。活性半胱天冬酶-1 切割促炎细胞因子 IL-1β 和 IL-18 以及形成孔的蛋白 GSDMD（gasdermin-D），从而释放其 N 端结构域。GSDMD N 端在质膜上形成多聚体孔，允许细胞内内容物泄漏，最终引发一种称为“细胞焦亡”的细胞死亡类型。新出现的研究表明，GSDMD 也被凋亡半胱天冬酶-8/-3/-7 加工。在本章中，我们旨在描述监测裂解性细胞死亡的方法，并区分 GSDMD 加工事件与细胞焦亡或凋亡诱导后产生的 GSDMD 片段。我们还说明了 GSDMD 孔形成与最终细胞裂解之间的差异，以及这如何影响细胞内物质的释放。最后，我们表明另一种形成孔的蛋白 gasdermin-E 的激活并不一定会导致骨髓来源的巨噬细胞发生裂解性细胞死亡。
