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. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.

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. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.

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.

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 pyroptosis1,2. Whereas caspase-1 processes IL-1ß and IL-18 for maturation3-6, no cytokine target has been firmly established for lipopolysaccharide-activated caspase-4/5/117-9. 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. Caspase-4 cleaves the same tetrapeptide site in pro-IL-18 as caspase-1. The crystal structure of the caspase-4-pro-IL-18 complex reveals a two-site (binary) substrate-recognition mechanism; the catalytic pocket engages the tetrapeptide, and a unique exosite that critically recognizes GSDMD10 similarly binds to a specific structure formed jointly by the propeptide and post-cleavage-site sequences in pro-IL-18. This binary recognition is also used by caspase-5 as well as caspase-1 to process pro-IL-18. In caspase-11, a structural deviation around the exosite underlies its inability to target pro-IL-18, which is restored by rationally designed mutations. The structure of pro-IL-18 features autoinhibitory interactions between the propeptide and the post-cleavage-site region, preventing recognition by the IL-18Rα receptor. Cleavage by caspase-1, -4 or -5 induces substantial conformational changes of IL-18 to generate two critical receptor-binding sites. Our study establishes IL-18 as a target of lipopolysaccharide-activated caspase-4/5. The finding is paradigm shifting in the understanding of noncanonical-inflammasome-mediated defences and also the function of IL-18 in immunity and disease.

Structural mechanisms for regulation of GSDMB pore-forming activity.

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein1,2, to trigger target cell pyroptosis3. GSDMB and the charter gasdermin family member GSDMD4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently6,8. Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins9,10. GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies6,8. Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms.

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 GSDMD1-5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.

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 Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

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 pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.

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 signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, 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-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.

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. GSDMD belongs to a gasdermin family that shares the pore-forming domain. The functions and mechanisms of activation of other gasdermins are unknown. Here we show that GSDME, which was originally identified as DFNA5 (deafness, autosomal dominant 5), can switch caspase-3-mediated apoptosis induced by TNF or chemotherapy drugs to pyroptosis. GSDME was specifically cleaved by caspase-3 in its linker, generating a GSDME-N fragment that perforates membranes and thereby induces pyroptosis. After chemotherapy, cleavage of GSDME by caspase-3 induced pyroptosis in certain GSDME-expressing cancer cells. GSDME was silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibited GSDME-dependent pyroptosis upon activation of caspase-3 by chemotherapy drugs. Gsdme-/- (also known as Dfna5-/-) mice were protected from chemotherapy-induced tissue damage and weight loss. These findings suggest that caspase-3 activation can trigger necrosis by cleaving GSDME and offer new insights into cancer chemotherapy.

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. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10­14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.

Inflammatory caspases are innate immune receptors for intracellular LPS.

The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation.

Structural insights into the unique single-stranded DNA-binding mode of Helicobacter pylori DprA.

Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to 'open' the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA-ssDNA.

Biochemical and structural insights into RNA binding by Ssh10b, a member of the highly conserved Sac10b protein family in Archaea.

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular ß-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.

Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins.

Cephalosporins constitute a large class of ß-lactam antibiotics clinically used as antimicrobial drugs. New Dehli metallo-ß-lactamase (NDM-1) poses a global threat to human health as it confers on bacterial pathogen resistance to almost all ß-lactams, including penicillins, cephalosporins, and carbapenems. Here we report the first crystal structures of NDM-1 in complex with cefuroxime and cephalexin, as well as NMR spectra monitoring cefuroxime and cefixime hydrolysis catalyzed by NDM-1. Surprisingly, cephalosporoate intermediates were captured in both crystal structures determined at 1.3 and 2.0 Å. These results provide detailed information concerning the mechanism and pathways of cephalosporin hydrolysis. We also present the crystal structure and enzyme assays of a D124N mutant, which reveals that D124 most likely plays a more structural than catalytic role.

Dissecting caspase-2-mediated cell death: from intrinsic PIDDosome activation to chemical modulation.

Caspase-2, a highly conserved member of the caspase family, is considered an initiator caspase that triggers apoptosis in response to some cellular stresses. Previous studies suggest that an intracellular multi-protein complex PIDDosome, induced by genotoxic stress, serves as a platform for caspase-2 activation. However, due to caspase-2's inability to process effector caspases, the mechanism underlying caspase-2-mediated cell death upon PIDDosome activation remains unclear. Here we conducted an unbiased genome-wide genetic screen and identified that the Bcl2 family protein BID is required for PIDDosome-induced, caspase-2-mediated apoptosis. PIDDosome-activated caspase-2 directly and functionally processes BID to signal the mitochondrial pathway for apoptosis induction. Additionally, a designed chemical screen identified a compound, HUHS015, that specifically activates caspase-2-mediated apoptosis. HUHS015-stimulated apoptosis also requires BID but is independent of the PIDDosome. Through extensive structure-activity relationship efforts, we identified a derivative with a potency of ~ 60 nmol/L in activating caspase-2-mediated apoptosis. The HUHS015-series of compounds act as efficient agonists that directly target the interdomain linker in caspase-2, representing a new mode of initiator caspase activation. Human and mouse caspase-2 differ in two crucial residues in the linker, rendering a selectivity of the agonists for human caspase-2. The caspase-2 agonists are valuable tools to explore the physiological roles of caspase-2-mediated cell death and a base for developing small-molecule drugs for relevant diseases.

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. First, TrichoGSDM, a pore-forming domain-only protein from the basal metazoan Trichoplax adhaerens, is a disulfides-linked autoinhibited dimer activated by reduction of the disulfides. The cryo-electron microscopy (cryo-EM) structure illustrates the assembly mechanism for the 44-mer TrichoGSDM pore. Second, RCD-1-1 and RCD-1-2, encoded by the polymorphic regulator of cell death-1 (rcd-1) gene in filamentous fungus Neurospora crassa, are also pore-forming domain-only GSDMs. RCD-1-1 and RCD-1-2, when encountering each other, form pores and cause pyroptosis, underlying allorecognition in Neurospora. The cryo-EM structure reveals a pore of 11 RCD-1-1/RCD-1-2 heterodimers and a heterodimerization-triggered pore assembly mechanism. This study shows mechanistic diversities in GSDM activation and indicates versatile functions of GSDMs.

Structural insights into the Pseudomonas aeruginosa type VI virulence effector Tse1 bacteriolysis and self-protection mechanisms.

Recently, it was identified that Pseudomonas aeruginosa competes with rival cells to gain a growth advantage using a novel mechanism that includes two interrelated processes as follows: employing type VI secretion system (T6SS) virulence effectors to lyse other bacteria, and at the same time producing specialized immunity proteins to inactivate their cognate effectors for self-protection against mutual toxicity. To explore the structural basis of these processes in the context of functional performance, the crystal structures of the T6SS virulence effector Tse1 and its complex with the corresponding immunity protein Tsi1 were determined, which, in association with mutagenesis and Biacore analyses, provided a molecular platform to resolve the relevant structural questions. The results indicated that Tse1 features a papain-like structure and conserved catalytic site with distinct substrate-binding sites to hydrolyze its murein peptide substrate. The immunity protein Tsi1 interacts with Tse1 via a unique interactive recognition mode to shield Tse1 from its physiological substrate. These findings reveal both the structural mechanisms for bacteriolysis and the self-protection against the T6SS effector Tse1. These mechanisms are significant not only by contributing to a novel understanding of niche competition among bacteria but also in providing a structural basis for antibacterial agent design and the development of new strategies to fight P. aeruginosa.