Shiyu Xia1, Jianbin Ruan2, Hao Wu3. 1. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States. 2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States. Electronic address: jianbin.ruan@childrens.harvard.edu. 3. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, United States; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States. Electronic address: wu@crystal.harvard.edu.
Abstract
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.
The pan class="Gene">gasdermin (n>n class="Gene">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 recombinantGSDMs 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.