Monitoring gasdermin pore formation in vitro.
The gasdermin (GSDM) family consists of gasdermin A (GSDMA), B (GSDMB), C (GSDMC), D (GSDMD), E or DNFA5 (GSDME), and DFNB59 in human. Expressed in the skin, gastrointestinal tract, and various immune cells, GSDMs mediate homeostasis and inflammation upon activation by caspases and unknown proteases. In particular, GSDMD is activated by inflammasome-activated caspases-1/-4/-5/-11 as well as a caspase-8-mediated pathway during Yersinia infection. These caspases cleave GSDMD to release its functional N-terminal fragment (GSDMD-NT) from its auto-inhibitory C-terminal fragment (GSDMD-CT). GSDMD-NTs bind to acid lipids in mammalian cell membranes and bacterial membranes, oligomerize, and insert into the membranes to form large transmembrane pores. Consequently, cellular contents including inflammatory cytokines are released and cells can undergo pyroptosis, a highly inflammatory form of cell death. In this chapter, we summarize recent research findings and present experimental procedures to obtain pure recombinant GSDMs for biochemical studies. We highlight a liposome-based assay that yields robust fluorescence signals for characterizing GSDM activities in vitro and may be applicable to other pore-forming proteins and ion channels in general.
['Xia|Shiyu|S|', 'Ruan|Jianbin|J|', 'Wu|Hao|H|']
D000818:Animals D014408:Biomarkers, Tumor D020169:Caspases D004268:DNA-Binding Proteins D006801:Humans D058847:Inflammasomes D007249:Inflammation D047908:Intracellular Signaling Peptides and Proteins D008081:Liposomes D009363:Neoplasm Proteins D009419:Nerve Tissue Proteins D028044:Phosphate-Binding Proteins D000069292:Pyroptosis D011960:Receptors, Estrogen
Gasdermin Immunity Inflammation Caspases Pyroptosis Pore-forming protein Liposome