Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation

Corey Rogers,Dan A Erkes,Emad S Alnemri,Teresa Fernandes-Alnemri,Andrew E Aplin,Alexandria Nardone

doi:10.1038/s41467-019-09397-2

Corey Rogers, Dan A Erkes + Show 4 more

Open Access

https://doi.org/10.1038/s41467-019-09397-2

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Abstract

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.

Highlights

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane
The intrinsic pathway is activated by internal stress arising from stimuli such as DNA damage, viral infection, glucocorticoids, and hypoxia leading to Bax/Bak-mediated pore formation on the outer mitochondrial membrane, which facilitates the release of proapoptotic proteins such as cytochrome c (Cyt c) and HtrA2/ Omi into the cytosol[3,4]
Deletion of GSDME considerably decreased the kinetics and magnitude of caspase-3 activation by triamcinolone acetonide (TA) treatment (Fig. 1d, h). These effects were not specific to TA treatment or to CEM-C7 cells but were observed with other apoptotic stimuli in CEM-C7 cells (Fig. 1e–h, Supplementary Fig. 1d–f), and in other cell types such as immortalized bone marrow-derived macrophages (Supplementary Fig. 2), the murine melanoma cell line B16 (Supplementary Fig. 3), and the murine thymoma cell line EG7-Ova (Supplementary Fig. 4). These results show that GSDME functions both downstream of caspase-3 to induce pyroptosis and upstream of caspase-3 to augment caspase-3 activation

Summary

Introduction

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Apoptosis is a form of programmed cell death (PCD) that plays critical roles in embryonic development, maintenance and regulation of a healthy immune system, and tumor suppression It is initiated in cells by a diverse range of physiological and pathological stimuli, which lead to activation of the intrinsic or extrinsic apoptotic pathways[1,2]. GSDMA, GSDMB, and GSDMC possess poreforming gasdermin-N domains[12], but none of them have been shown to be cleaved in response to physiological or pathological stimuli to form functional pores In addition to their necrotic activity, GSDMA, GSDMC, GSDMD, and GSDME have all been proposed to possess tumor suppressive activity, as their expression suppresses cell proliferation and colony formation in gastric and colorectal cancer cell lines[17,18,19,20]. GSDME expression is regulated by p5324, which is known to activate the transcription of numerous tumor suppressors and activators of apoptosis

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