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Published on: May 21, 2018
Gasdermin D Cleavage Assay Following Inflammasome Activation
Louisa Janice Kamajaya1, Dave Boucher2
1Department of Biology, York Biomedical Research Institute, University of York, Heslington, York, UK.
Abstract:
Gasdermin D (GSDMD) is a recently identified pore-forming protein that is crucial for the execution of pyroptosis, a highly inflammatory form of cell death. GSDMD contains an N-terminal and a C-terminal domain that are separated by a proteolysis-sensitive linker. Upon cleavage of this linker by inflammasome-activated caspases, the N-terminal domain of GSDMD oligomerizes and forms pores at the plasma membrane, allowing cell swelling and subsequently membrane rupture to mediate pyroptosis. GSDMD is a key substrate of inflammatory caspases downstream of inflammasome activation and is driving various pathologies. Here, we describe a simple method to study GSDMD cleavage following canonical inflammasome activation in murine primary macrophages and neutrophils and human cell lines using immunoblotting.
Insights
Gasdermin D (GSDMD) cleavage initiates pyroptosis, a cell death pathway. This study presents a simple immunoblotting method to analyze GSDMD cleavage in immune cells following inflammasome activation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Gasdermin D (GSDMD) is a key protein in pyroptosis, a pro-inflammatory cell death.
- GSDMD forms pores in the cell membrane after cleavage by caspases.
- Dysregulation of GSDMD is implicated in various diseases.
Purpose of the Study:
- To develop and present a straightforward method for studying Gasdermin D (GSDMD) cleavage.
- To investigate GSDMD cleavage in response to canonical inflammasome activation.
Main Methods:
- Utilized immunoblotting techniques.
- Studied GSDMD cleavage in murine primary macrophages and neutrophils.
- Examined GSDMD cleavage in human cell lines.
Main Results:
- Successfully established a simple method to detect GSDMD cleavage.
- Demonstrated the method's applicability in different cell types and following inflammasome activation.
Conclusions:
- The described immunoblotting method is effective for studying GSDMD cleavage.
- This technique facilitates research into pyroptosis and associated pathologies.
