Live-cell visualization of gasdermin D-driven pyroptotic cell death.

Autor:	Rathkey JK; From the Department of Pathology., Benson BL; From the Department of Pathology.; the Division of Pediatric Hematology-Oncology, Department of Pediatrics, and., Chirieleison SM; From the Department of Pathology., Yang J; From the Department of Pathology.; the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106., Xiao TS; From the Department of Pathology., Dubyak GR; the Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106., Huang AY; From the Department of Pathology.; the Division of Pediatric Hematology-Oncology, Department of Pediatrics, and., Abbott DW; From the Department of Pathology, dwa4@case.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2017 Sep 01; Vol. 292 (35), pp. 14649-14658. Date of Electronic Publication: 2017 Jul 18.
DOI:	10.1074/jbc.M117.797217
Abstrakt:	Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization. (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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