Assembly, purification, and assay of the activity of the ASC pyroptosome.
Pyroptosis is an inflammatory form of cell death mediated by caspase-1. Until recently, little was known about the mechanism by which caspase-1 is specifically activated to induce pyroptosis. Using biochemical and time-lapse confocal bioimaging approaches, it has been shown that caspase-1 is activated during pyroptosis by a large supramolecular assembly termed the pyroptosome. Biochemical and mass spectroscopic analyses revealed that the pyroptosome assembly is an oligomer of dimers of the adaptor protein ASC. Only one distinct pyroptosome is formed in each cell when macrophages or monocytes are stimulated with proinflammatory stimuli, which rapidly recruits and activates caspase-1, resulting in pyroptosis. This chapter describes methods for real-time observation and recording of the pyroptosome assembly process in live THP-1 monocytes. It also describes biochemical methods for the assembly, purification, and assay of the ASC pyroptosome from the THP-1 cell line, which could be adapted for use with other cell lines containing ASC, such as primary mouse macrophages. Finally, it describes methods for the in vitro reconstitution of a functional ASC pyroptosome from the recombinant ASC protein produced in Escherichia coli.
Fernandes-Alnemri
Teresa
T
Department of Biochemistry and Molecular Biology, Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Alnemri
Emad S
ES
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D020170:Caspase 1 / Q000378:metabolism*; D016923:Cell Death / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence
D048868:Adaptor Proteins, Signal Transducing; D049452:Green Fluorescent Proteins; D020170:Caspase 1