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The kinase Mst1, which acts in the Hippo pathway, controls cell proliferation, differentiation and apoptosis. Junichi Sadoshima and his colleagues show that Mst1 in cardiomyocytes phosphorylates the protein Beclin1 to coordinately suppress autophagy and promote apoptosis, thereby having deleterious effects on the heart. Here we show that Mst1, a proapoptotic kinase, impairs protein quality control mechanisms in the heart through inhibition of autophagy. Stress-induced activation of Mst1 in cardiomyocytes promoted accumulation of p62 and aggresome formation, accompanied by the disappearance of autophagosomes. Mst1 phosphorylated the Thr108 residue in the BH3 domain of Beclin1, which enhanced the interaction between Beclin1 and Bcl-2 and/or Bcl-xL, stabilized the Beclin1 homodimer, inhibited the phosphatidylinositide 3-kinase activity of the Atg14L-Beclin1-Vps34 complex and suppressed autophagy. Furthermore, Mst1-induced sequestration of Bcl-2 and Bcl-xL by Beclin1 allows Bax to become active, thereby stimulating apoptosis. Mst1 promoted cardiac dysfunction in mice subjected to myocardial infarction by inhibiting autophagy, associated with increased levels of Thr108-phosphorylated Beclin1. Moreover, dilated cardiomyopathy in humans was associated with increased levels of Thr108-phosphorylated Beclin1 and signs of autophagic suppression. These results suggest that Mst1 coordinately regulates autophagy and apoptosis by phosphorylating Beclin1 and consequently modulating a three-way interaction among Bcl-2 proteins, Beclin1 and Bax.

Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D ( Gsdmd ) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd −/− cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis. CRISPR-Cas9 genome-editing screens identify gasdermin D as a substrate for inflammatory caspases, and its N-terminal cleavage fragment, as well as the equivalent regions in other gasdermins, is shown to be capable of inducing pyroptosis. Gasdermin-D role in innate immunity Two groups reporting in this issue of Nature identify gasdermin D, the product of the Gsdmd gene conserved in human and mouse but of unknown function, as a substrate for inflammatory caspases. Feng Shao and co-workers use genome-wide CRISPR–Cas9 screens to identify gasdermin D as a substrate for inflammatory caspases. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains. Vishva Dixit and co-workers use an ENU mutagenesis screen to identify gasdermin D as the required substrate for pyroptosis-mediating caspase-11 in the non-canonical inflammasome pathway. Mice lacking gasdermin D are protected from a lethal dose of lipopolysaccharide. Both groups show that the cleaved N-terminal domain is sufficient to trigger pyroptosis, a form of programmed necrotic cell death.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli , Salmonella typhimurium , Shigella flexneri , and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl- N -nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd −/− mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd −/− mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria. Gasdermin D is identified as the required substrate for pyroptosis, mediating caspase-11 function in the non-canonical inflammasome pathway; the cleaved N-terminal domain is shown to trigger pyroptosis.

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium -treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd −/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd −/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.

A classic feature of apoptotic cells is the cell-surface exposure of phosphatidylserine (PtdSer) as an \"eat me\" signal for engulfment. We show that the Xk-family protein Xkr8 mediates PtdSer exposure in response to apoptotic stimuli. Mouse Xkr8 -/- cells or human cancer cells in which Xkr8 expression was repressed by hypermethylation failed to expose PtdSer during apoptosis and were inefficiently engulfed by phagocytes. Xkr8 was activated directly by caspases and required a caspase-3 cleavage site for its function. CED-8, the only Caenorhabditis elegans Xk-family homolog, also promoted apoptotic PtdSer exposure and cell-corpse engulfment. Thus, Xk-family proteins have evolutionarily conserved roles in promoting the phagocytosis of dying cells by altering the phospholipid distribution in the plasma membrane.

The lysosomal degradation pathway of autophagy has a crucial role in defence against infection, neurodegenerative disorders, cancer and ageing. Accordingly, agents that induce autophagy may have broad therapeutic applications. One approach to developing such agents is to exploit autophagy manipulation strategies used by microbial virulence factors. Here we show that a peptide, Tat–beclin 1—derived from a region of the autophagy protein, beclin 1, which binds human immunodeficiency virus (HIV)-1 Nef—is a potent inducer of autophagy, and interacts with a newly identified negative regulator of autophagy, GAPR-1 (also called GLIPR2). Tat–beclin 1 decreases the accumulation of polyglutamine expansion protein aggregates and the replication of several pathogens (including HIV-1) in vitro , and reduces mortality in mice infected with chikungunya or West Nile virus. Thus, through the characterization of a domain of beclin 1 that interacts with HIV-1 Nef, we have developed an autophagy-inducing peptide that has potential efficacy in the treatment of human diseases. A cell-permeable peptide is constructed that is derived from a region of an essential autophagy protein called beclin 1; the peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice, and is effective in the clearance of several viruses. Autophagy inducer with potential Autophagy is an essential degradation pathway that eliminates damaged proteins and organelles in cells and also protects against infection by diverse pathogens, including viruses. In this study, Beth Levine and colleagues construct a cell-permeable peptide, Tat-beclin 1, derived from part of an essential autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice, and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1. The Tat-beclin 1 peptide binds to the Golgi-associated plant pathogenesis-related protein 1 (GAPR-1), which functions as a negative regulator of autophagy. These results suggest that this beclin 1-derived autophagy-inducing peptide has potential for the prevention and treatment of a broad range of human diseases.

Resistance represents a major challenge for antibody-based therapy for COVID-19 1 , 2 , 3 – 4 . Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern—B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)—and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19. An engineered IgM antibody administered intranasally in mice shows high prophylactic efficacy and therapeutic efficacy against SARS-CoV-2, and is also effective against multiple variants of concern that are resistant to IgG-based therapeutics.

NEK7, a member of the NIMA-related kinase family, is identified as a regulator of NLRP3 inflammasome oligomerization and activation; NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. NEK7 mediates NLRP3 inflammasome activation The NLRP3 inflammasome, a critical component of the innate immune system, has been linked to multiple acquired and inherited diseases. However, the molecular mechanism that leads to NLRP3 oligomerization and activation remains elusive. Here Gabriel Núñez and colleagues identify a member of the family of NIM related kinases (NEK7) as a regulator of NLRP3 inflammasome oligomerization and activation. NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases 1 . So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins 2 , 3 . The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases 4 , 5 as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations 6 , 7 . Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli 8 , 9 . Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins) 10 , as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3–NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3–NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7 −/− or Nlrp3 −/− haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo . These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form amultiprotein complex called an inflammasome. Here we report the cryo–electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types. In inflammasomes, caspase-1 activation leads to pyroptosis mediated by gasdermin D, but cells lacking gasdermin-D still initiate caspase-dependent cell death. Here, Tsuchiya et al. show that these cells undergo Bid- and caspase-3-dependent apoptosis.