GSDMD基因敲除HEK293细胞

BACKGROUND AND AIMS:Enhanced inflammatory signalling causally contributes to atrial fibrillation (AF) development. Gasdermin D (GSDMD) is an important downstream effector of several inflammasome pathways. However, the role of GSDMD, particularly the cleaved N-terminal (NT)-GSDMD, in non-immune cells remains elusive. This study aimed to elucidate the function of NT-GSDMD in atrial cardiomyocytes (ACMs) and determine its contribution to atrial arrhythmogenesis. METHODS:Human atrial appendages were used to assess the protein levels and localization. A modified adeno-associated virus 9 was employed to establish ACM-restricted overexpression of NT-GSDMD in mice. RESULTS:The cleavage of GSDMD was enhanced in ACMs of AF patients. Atrial cardiomyocyte-restricted overexpression of NT-GSDMD in mice increased susceptibility to pacing-induced AF. The NT-GSDMD pore formation facilitated interleukin-1β secretion from ACMs, promoting macrophage infiltration, while up-regulating 'endosomal sorting complexes required for transport'-mediated membrane-repair mechanisms, which prevented inflammatory cell death (pyroptosis) in ACMs. Up-regulated NT-GSDMD directly targeted mitochondria, increasing mitochondrial reactive oxygen species (ROS) generation, which triggered proarrhythmic calcium-release events. The NT-GSDMD-induced arrhythmogenesis was mitigated by the mitochondrial-specific antioxidant MitoTEMPO. A mutant NT-GSDMD lacking pore-formation capability failed to cause mitochondrial dysfunction or induce atrial arrhythmia. Genetic ablation of Gsdmd prevented spontaneous AF development in a mouse model. CONCLUSIONS:These findings establish a unique pyroptosis-independent role of NT-GSDMD in ACMs and arrhythmogenesis, which involves ROS-driven mitochondrial dysfunction. Mitochondrial-targeted therapy, either by reducing ROS production or inhibition of GSDMD, prevents AF inducibility, positioning GSDMD as a novel therapeutic target for AF prevention.

The NLRP3 inflammasome plays a critical role in innate immunity and inflammatory diseases. NIMA-related kinase 7 (NEK7) is essential for inflammasome activation, and its interaction with NLRP3 is enhanced by K efflux. However, the mechanism by which K efflux promotes this interaction remains unknown. Here, we show that NEK7 is rapidly phosphorylated at threonine-190/191 by JNK1 downstream of K efflux and gasdermin D (GSDMD) after NLRP3 activation. NEK7 phosphorylation enhances the binding between NEK7 and NLRP3, which further promotes inflammasome assembly and activation. Mutant mice and macrophages in which Thr and Thr of Nek7 were replaced by valine exhibited impaired NEK7 phosphorylation, NLRP3 inflammasome activation, and IL-1β secretion. Thus, NEK7 phosphorylation is an important event that acts downstream of K efflux and GSDMD to further enhance NLRP3 inflammasome activation.

RING1 is an E3 ligase component of the polycomb repressive complex 1 (PRC1) with known roles in chromatin regulation and cellular processes such as apoptosis and autophagy. However, its involvement in inflammation and pyroptosis remains elusive. Here, we demonstrate that human RING1, not RING2, promotes K48-linked ubiquitination of Gasdermin D (GSDMD) and acts as a negative regulator of pyroptosis and bacterial infection. Indeed, we showed that loss of Ring1 increased S. typhimurium infectious load and mortality in vivo. Though RING1 deletion initially reduced M. tuberculosis (Mtb) infectious load in vivo, increased lung inflammation and impaired immune defense responses were later observed. Moreover, Ring1 knockout exacerbated acute sepsis induced by lipopolysaccharide (LPS) in vivo. Mechanistically, RING1 directly interacts with GSDMD and ubiquitinates the K51 and K168 sites of GSDMD for K48-linked proteasomal degradation, thereby inhibiting pyroptosis. Inhibition of RING1 E3 ligase activity by direct mutation or with the use of small molecule inhibitors increased GSDMD level and cell death during pyroptosis. Our findings reveal that RING1 dictates GSDMD-mediated inflammatory response and host susceptibility to pathogen infection, highlighting RING1 as a potential therapeutic target for combating infectious diseases.

SARS-CoV-2 3C-like protease (3CL or M) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL expression and viral replication. Unexpectedly, 3CL and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CL, which decreases in GsdmdGsdme mice. We identify new 3CL cut-sites in GSDMD at LQ↓SS, which blocks pore formation by 3CL cleavage at LH↓N lying adjacent to the caspase activation site (NFLTD↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CL secretion. Extracellular 3CL retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-λ1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CL is secreted with extracellular pathological sequelae.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.