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Protein engineering reveals that gasdermin A preferentially targets mitochondrial membranes over the plasma membrane
Hannah C Kondolf1, Dana A D'Orlando1, George R Dubyak2
1Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.
Abstract:
When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.
Insights
Gasdermin family proteins cause cell death, but their activation mechanisms differ. This study reveals distinct cell death pathways for gasdermin D (GSDMD) and gasdermin A (GSDMA), impacting immunological outcomes.
Area of Science:
- Cellular Biology
- Immunology
- Molecular Mechanisms of Cell Death
Background:
- Gasdermin proteins are key mediators of lytic cell death, including pyroptosis.
- The precise mechanisms and activation thresholds for different gasdermin family members remain unclear.
- Previous studies suggest varying propensities for pyroptosis among gasdermins.
Purpose of the Study:
- To develop a standardized system for activating gasdermin family members.
- To investigate and compare the subcellular localization and cell death-inducing capabilities of GSDMD and GSDMA.
- To challenge the assumption of identical cell death mechanisms among gasdermins.
Main Methods:
- Utilized a novel system for efficient and equivalent activation of gasdermin family members.
- Leveraged exosite-mediated recognition of GSDMD by inflammatory caspases for activation.
- Compared subcellular localization, plasma membrane permeabilization, and cell death kinetics of GSDMD and GSDMA.
Main Results:
- GSDMD rapidly localized to plasma and organelle membranes, while GSDMA preferentially localized to mitochondria.
- GSDMA showed delayed and diminished accumulation at the plasma membrane compared to GSDMD.
- N-terminal GSDMA induced early mitochondrial dysfunction preceding plasma membrane permeabilization.
Conclusions:
- Gasdermin family members exhibit differential subcellular localization and cell death kinetics upon activation.
- GSDMD and GSDMA mediate distinct cell death pathways, challenging the notion of uniform mechanisms.
- Differential gasdermin activation likely results in varied immunological outcomes in specific tissues.
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