Cleavage-independent activation of ancient eukaryotic gasdermins and structural mechanisms.

Gasdermins (GSDMs) are pore-forming proteins that execute pyroptosis for immune defense. GSDMs are two-domain proteins activated by proteolytic removal of the inhibitory domain. In this work, we report two types of cleavage-independent GSDM activation.

Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome.

The canonical (caspase-1) and noncanonical (comprising caspases 4, 5 and 11; hereafter, caspase-4/5/11) inflammasomes both cleave gasdermin D (GSDMD) to induce pyroptosis. Whereas caspase-1 processes IL-1β and IL-18 for maturation, no cytokine target has been firmly established for lipopolysaccharide-activated caspase-4/5/11. Here we show that activated human caspase-4, but not mouse caspase-11, directly and efficiently processes IL-18 in vitro and during bacterial infections.

V-ATPase recruitment to ER exit sites switches COPII-mediated transport to lysosomal degradation.

Endoplasmic reticulum (ER)-phagy is crucial to regulate the function and homeostasis of the ER via lysosomal degradation, but how it is initiated is unclear. Here we discover that Z-AAT, a disease-causing mutant of α1-antitrypsin, induces noncanonical ER-phagy at ER exit sites (ERESs). Accumulation of misfolded Z-AAT at the ERESs impairs coat protein complex II (COPII)-mediated ER-to-Golgi transport and retains V0 subunits that further assemble V-ATPase at the arrested ERESs.

Determination of Gasdermin Pores.

The gasdermin family represents a type of membrane pore-forming proteins. The gasdermin family is extensively characterized as the executioner of pyroptotic cell death in mammals; recent studies suggest that gasdermin-like pore-forming proteins are also present in bacteria and fungi. In humans, gasdermin D (GSDMD) is activated through inter-domain cleavage by caspase-1 in the canonical inflammasome pathway and cytosolic LPS-activated caspase-4 or caspase-5.

Structural mechanisms for regulation of GSDMB pore-forming activity.

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein, to trigger target cell pyroptosis. GSDMB and the charter gasdermin family member GSDMD have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs.

Structural mechanisms of calmodulin activation of Shigella effector OspC3 to ADP-riboxanate caspase-4/11 and block pyroptosis.

The caspase-4/11-GSDMD pyroptosis axis recognizes cytosolic lipopolysaccharide for antibacterial defenses. Shigella flexneri employs an OspC3 effector to block pyroptosis by catalyzing NAD-dependent arginine ADP-riboxanation of caspase-4/11. Here, we identify Ca-free calmodulin (CaM) that binds and stimulates OspC3 ADP-riboxanase activity.

An anti-CD98 antibody displaying pH-dependent Fc-mediated tumour-specific activity against multiple cancers in CD98-humanized mice.

The cell-surface glycoprotein CD98-a subunit of the LAT1/CD98 amino acid transporter-is an attractive target for cancer immunotherapies, but its widespread expression has hampered the development of CD98-targeting antibody therapeutics. Here we report that an anti-CD98 antibody, identified via the screening of phage-display libraries of CD98 single-chain variable fragments with mutated complementarity-determining regions, preserves the physiological function of CD98 and elicits broad-spectrum crystallizable-fragment (Fc)-mediated anti-tumour activity (requiring Fcγ receptors for immunoglobulins, macrophages, dendritic cells and CD8 T cells, as well as other components of the innate and adaptive immune systems) in multiple xenograft and syngeneic tumour models established in CD98-humanized mice. We also show that a variant of the anti-CD98 antibody with pH-dependent binding, generated by solving the structure of the antibody-CD98 complex, displayed enhanced tumour-specific activity and pharmacokinetics.

Calmodulin Binding Activates CopC Effector to ADP-Riboxanate Host Apoptotic Caspases.

Blocking host cell death is an important virulence strategy employed by many bacterial pathogens. We recently reported that Shigella flexneri inhibits host pyroptosis by delivering a type III secretion system (T3SS) effector OspC3 that catalyzes a novel arginine ADP-riboxanation modification on caspase-4/11. Here, we investigated the OspC3 homologue CopC from Chromobacterium violaceum, an opportunistic but sometimes deadly bacterial pathogen.

Endomembrane damage sensing by V-ATPase recruits ATG16L1 for LC3 lipidation .

LC3 lipidation-mediated selective macroautophagy/autophagy helps eukaryotes to defend against endogenous dangers and foreign invaders. However, LC3 activation mechanisms of selective autophagy are still elusive. We previously determined that the V-ATPase-ATG16L1 axis is critical for LC3 recruitment to bacteria-residing vacuoles, whereas the effector SopF directly targets V-ATPase to disrupt ATG16L1 interaction.

ARF GTPases activate Salmonella effector SopF to ADP-ribosylate host V-ATPase and inhibit endomembrane damage-induced autophagy.

Selective autophagy helps eukaryotes to cope with endogenous dangers or foreign invaders; its initiation often involves membrane damage. By studying a Salmonella effector SopF, we recently identified the vacuolar ATPase (V-ATPase)-ATG16L1 axis that initiates bacteria-induced autophagy. Here we show that SopF is an ADP-ribosyltransferase specifically modifying Gln124 of ATP6V0C in V-ATPase.

Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11.

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD. This non-canonical inflammasome defends against Gram-negative bacteria. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside.

Structures of heat shock factor trimers bound to DNA.

Heat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular proteostasis or mediating cell differentiation and development. Upon activation, both HSFs trimerize and bind to heat shock elements (HSEs) present in the promoter region of target genes. Despite structural insights gained from recent studies, structures reflecting the physiological architecture of this transcriptional machinery remains to be determined.

Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.

Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)-positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins.

A bioorthogonal system reveals antitumour immune function of pyroptosis.

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown.

Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown.

A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.

Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy.

Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn as well as NleB-GlcNAcylated DDs of TRADD and RIPK1.

Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose.

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling. Recent studies indicate that the bacterial metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-β-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression.

Growing a gasdermin pore in membranes of pyroptotic cells.

Inflammasome‐activated caspase‐1, caspase‐11, caspase‐4, and caspase‐5 cleave GSDMD to unleash its N‐terminal gasdermin‐N domain (GSDMD ) that perforates the plasma membrane to execute pyroptosis and stimulate inflammation. The mechanism underlying GSDMD pore formation is unclear. Mulvihill use high‐resolution atomic force microscopy (AFM) to analyze the dynamic pore formation process of GSDMD .

Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.

Pyroptosis is a form of cell death that is critical for immunity. It can be induced by the canonical caspase-1 inflammasomes or by activation of caspase-4, -5 and -11 by cytosolic lipopolysaccharide. The caspases cleave gasdermin D (GSDMD) in its middle linker to release autoinhibition on its gasdermin-N domain, which executes pyroptosis via its pore-forming activity.

SnapShot: The Noncanonical Inflammasome.

This SnapShot depicts how the noncanonical inflammasome pathway is initiated and activated, as well as its effector mechanism in triggering pyroptosis and immune defenses.

Pore-forming activity and structural autoinhibition of the gasdermin family.

Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown.

Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori.

DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.