ABSTRACT
NLRP inflammasomes are a group of cytosolic multiprotein oligomer pattern recognition receptors (PRRs) involved in the recognition of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) produced by infected cells. They regulate innate immunity by triggering a protective inflammatory response. However, despite their protective role, aberrant NLPR inflammasome activation and gain-of-function mutations in NLRP sensor proteins are involved in occurrence and enhancement of non-communicating autoimmune, auto-inflammatory, and neurodegenerative diseases. In the last few years, significant advances have been achieved in the understanding of the NLRP inflammasome physiological functions and their molecular mechanisms of activation, as well as therapeutics that target NLRP inflammasome activity in inflammatory diseases. Here, we provide the latest research progress on NLRP inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRP7, NLRP2, NLRP9, NLRP10, and NLRP12 regarding their structural and assembling features, signaling transduction and molecular activation mechanisms. Importantly, we highlight the mechanisms associated with NLRP inflammasome dysregulation involved in numerous human auto-inflammatory, autoimmune, and neurodegenerative diseases. Overall, we summarize the latest discoveries in NLRP biology, their forming inflammasomes, and their role in health and diseases, and provide therapeutic strategies and perspectives for future studies about NLRP inflammasomes.
Subject(s)
Inflammasomes , NLR Proteins , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , NLR Proteins/metabolism , Animals , Neurodegenerative Diseases/immunology , Neurodegenerative Diseases/metabolism , Signal Transduction/immunology , Immunity, Innate , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , Inflammation/immunology , Inflammation/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/geneticsABSTRACT
During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1ß maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.
Subject(s)
Caspase 3 , Gasdermins , Intracellular Signaling Peptides and Proteins , Macrophages , Phosphate-Binding Proteins , Pyroptosis , Pyroptosis/drug effects , Phosphate-Binding Proteins/metabolism , Macrophages/metabolism , Macrophages/microbiology , Intracellular Signaling Peptides and Proteins/metabolism , Caspase 3/metabolism , Humans , Animals , Mice , Apoptosis Regulatory Proteins/metabolism , Bacterial Toxins/metabolism , Caspases/metabolism , Lipopolysaccharides/pharmacology , Enterohemorrhagic Escherichia coli/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Caspases, Initiator/metabolism , Inflammasomes/metabolism , Mice, Inbred C57BL , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Infections/immunology , Interleukin-1beta/metabolismABSTRACT
The gasdermins are a family of pore-forming proteins involved in various cellular processes such as cell death and inflammation. A new study in PLOS Biology explores the evolutionary history of gasdermins across metazoans, highlighting the conservation and divergence of gasdermin E.
Subject(s)
Lancelets , Pyroptosis , Animals , Pyroptosis/physiology , Lancelets/metabolism , Gasdermins , Neoplasm Proteins/metabolism , Defense MechanismsABSTRACT
Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis1,2. Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential3-7. Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity4. Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB4. Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB-IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity.
Subject(s)
Bacterial Proteins , Gasdermins , Pore Forming Cytotoxic Proteins , Animals , Humans , Mice , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Binding Sites , Conserved Sequence , Cryoelectron Microscopy , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/metabolism , Pore Forming Cytotoxic Proteins/ultrastructure , Protein Domains , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Isoforms/ultrastructure , Pyroptosis , Shigella , Species Specificity , Gasdermins/chemistry , Gasdermins/metabolism , Gasdermins/ultrastructureABSTRACT
The ripple effect induced by uncontrollable Zn deposition is considered as the Achilles heel for developing high-performance aqueous Zn-ion batteries. For this problem, this work reports a design concept of 3D artificial array interface engineering to achieve volume stress elimination, preferred orientation growth and dendrite-free stable Zn metal anode. The mechanism of MXene array interface on modulating the growth kinetics and deposition behavior of Zn atoms were firstly disclosed on the multi-scale level, including the in-situ optical microscopy and transient simulation at the mesoscopic scale, in-situ Raman spectroscopy and in-situ X-ray diffraction at the microscopic scale, as well as density functional theory calculation at the atomic scale. As indicated by the electrochemical performance tests, such engineered electrode exhibits the comprehensive enhancements not only in the resistance of corrosion and hydrogen evolution, but also the rate capability and cyclic stability. High-rate performance (20 mA cm-2) and durable cycle lifespan (1350 h at 0.5 mA cm-2, 1500 h at 1 mA cm-2 and 800 h at 5 mA cm-2) can be realized. Moreover, the improvement of rate capability (214.1 mAh g-1 obtained at 10 A g-1) and cyclic stability also can be demonstrated in the case of 3D MXene array@Zn/VO2 battery. Beyond the previous 2D closed interface engineering, this research offers a unique 3D open array interface engineering to stabilize Zn metal anode, the controllable Zn deposition mechanism revealed is also expected to deepen the fundamental of rechargeable batteries including but not limited to aqueous Zn metal batteries.
ABSTRACT
Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.
Subject(s)
CD18 Antigens , Neutrophils , Animals , CD18 Antigens/metabolism , Calcium/metabolism , Cell Adhesion , Guanosine , Guanosine Triphosphate/metabolism , Humans , Interleukin-8/metabolism , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , Polyphosphates , rac1 GTP-Binding Protein/metabolismABSTRACT
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
Subject(s)
Bacterial Toxins/metabolism , Enterococcus , Leukocytes, Mononuclear , Virulence Factors/metabolism , Animals , Cattle , Enterococcus/metabolism , Enterococcus/pathogenicity , Horses , Mice , Microbial Sensitivity Tests , SwineABSTRACT
The gasdermin family is a newly identified class of pore-forming proteins that play as the executioners of pyroptosis, a lytic pro-inflammatory type of cell death triggered by sensing cytosolic infections and danger signals. Upon activation, the gasdermin N-terminal domain translocates to the cell membrane to form pores, which allow the release of proinflammatory cytokines and alarmins, and cause cell lysis. Many structural studies have been conducted in the past few years to investigate the mechanisms of gasdermin proteins in the activation and pore formation. Here, we review these high-resolution structures and highlight the mechanistic insights into the gasdermin activation and regulation that are provided.
Subject(s)
Phosphate-Binding Proteins , Pore Forming Cytotoxic Proteins , Pyroptosis , Cell Membrane/metabolism , Cytokines/metabolism , Humans , Inflammasomes/metabolism , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Pyroptosis/physiologyABSTRACT
As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.
Subject(s)
Inflammasomes/chemistry , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Macrophages/metabolism , Phosphate-Binding Proteins/chemistry , Animals , Caspase 1/metabolism , Cryoelectron Microscopy , Humans , Interleukin-1/metabolism , Mice, Inbred C57BL , Protein Precursors/metabolism , Protein Structure, Quaternary , Static ElectricityABSTRACT
Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.
Subject(s)
Alarmins/metabolism , Endotoxemia/immunology , Galectin 1/metabolism , Inflammation Mediators/metabolism , Inflammation/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/metabolism , Phosphate-Binding Proteins/metabolism , Adult , Aged , Aged, 80 and over , Alarmins/deficiency , Alarmins/genetics , Animals , Case-Control Studies , Disease Models, Animal , Endotoxemia/chemically induced , Endotoxemia/metabolism , Endotoxemia/pathology , Female , Galectin 1/blood , Galectin 1/deficiency , Galectin 1/genetics , HeLa Cells , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/pathology , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Leukocyte Common Antigens/metabolism , Lipopolysaccharides , Macrophages/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Necroptosis , Phosphate-Binding Proteins/deficiency , Phosphate-Binding Proteins/genetics , RAW 264.7 Cells , Sepsis/blood , Sepsis/diagnosis , Signal Transduction , Up-RegulationABSTRACT
NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD-caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Ankyrins/metabolism , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins/metabolism , Inflammasomes/metabolism , Neoplasm Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Ankyrins/chemistry , Apoptosis , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , CARD Signaling Adaptor Proteins/chemistry , CARD Signaling Adaptor Proteins/genetics , Caspase 1/metabolism , Caspase Activation and Recruitment Domain , Cryoelectron Microscopy , Death Domain Receptor Signaling Adaptor Proteins/chemistry , Death Domain Receptor Signaling Adaptor Proteins/metabolism , HEK293 Cells , Humans , Inflammasomes/chemistry , Inflammasomes/ultrastructure , Models, Molecular , NLR Proteins , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Protein Interaction Domains and Motifs , Signal TransductionABSTRACT
Inflammatory caspase-dependent cytosolic lipopolysaccharide (LPS) sensing is a critical arm of host defense against bacteria. How pathogens overcome this pathway to establish infections is largely unknown. Enterohemorrhagic Escherichia coli (EHEC) is a clinically important human pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. We found that a bacteriophage-encoded virulence factor of EHEC, Shiga toxin (Stx), suppresses caspase-11-mediated activation of the cytosolic LPS sensing pathway. Stx was essential and sufficient to inhibit pyroptosis and interleukin-1 (IL-1) responses elicited specifically by cytosolic LPS. The catalytic activity of Stx was necessary for suppression of inflammasome responses. Stx impairment of inflammasome responses to cytosolic LPS occurs at the level of gasdermin D activation. Stx also suppresses inflammasome responses in vivo after LPS challenge and bacterial infection. Overall, this study assigns a previously undescribed inflammasome-subversive function to a well-known bacterial toxin, Stx, and reveals a new phage protein-based pathogen blockade of cytosolic immune surveillance.
Subject(s)
Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/immunology , Inflammasomes/immunology , Shiga Toxin 1/metabolism , Shiga Toxin 2/metabolism , Animals , Bacteriophages/immunology , Bacteriophages/metabolism , Caspases, Initiator/genetics , Caspases, Initiator/metabolism , Chlorocebus aethiops , Citrobacter rodentium/immunology , Citrobacter rodentium/pathogenicity , Disease Models, Animal , Enterohemorrhagic Escherichia coli/immunology , Enterohemorrhagic Escherichia coli/virology , Escherichia coli Infections/microbiology , Female , Humans , Immunologic Surveillance , Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/immunology , Male , Mice , Mice, Knockout , Phosphate-Binding Proteins/metabolism , Shiga Toxin 1/immunology , Shiga Toxin 2/immunology , Vero Cells , Viral Proteins/immunology , Viral Proteins/metabolism , Virulence Factors/immunology , Virulence Factors/metabolismABSTRACT
Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.
Subject(s)
Disulfiram/pharmacology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Phosphate-Binding Proteins/antagonists & inhibitors , Pyroptosis/drug effects , Sepsis/drug therapy , Animals , Caspase 1/genetics , Caspase 1/metabolism , Caspase Inhibitors/pharmacology , Caspases/metabolism , Caspases, Initiator/genetics , Caspases, Initiator/metabolism , Cell Line, Tumor , Disulfiram/therapeutic use , Drug Evaluation, Preclinical , Drug Repositioning , Female , HEK293 Cells , High-Throughput Screening Assays , Humans , Interleukin-1beta/immunology , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/immunology , Liposomes , Mice , Mutagenesis, Site-Directed , Phosphate-Binding Proteins/genetics , Phosphate-Binding Proteins/metabolism , Pyroptosis/immunology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sepsis/immunology , Sf9 Cells , SpodopteraABSTRACT
Gasdermin is a recently identified family of pore-forming proteins consisting of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C (GSDMC), Gasdermin D (GSDMD), Gasdermin E (GSDME), and DFNB59. Gasdermin D (GSDMD) is a downstream effector of inflammasomes, which are supramolecular complexes that activate inflammatory caspases (-1, -4, and -5 in human and -1 and -11 in mouse). GSDMD contains a functionally important N-terminal domain (GSDMD-N), a C-terminal domain, and a linker in between that is recognized and cleaved by the activated inflammatory caspases. Upon cleavage, the GSDMD-N fragments translocate on the membrane and oligomerize to form membrane-embedded pores after specifically binding to acidic lipids such as phosphatidylinositol phosphates (PIPs), phosphatidic acid (PA), phosphatidylserine (PS), and cardiolipin. The pore exhibits strong membrane-disrupting cytotoxicity in mammalian cells by disrupting the osmotic potential and also serves as a gate for extracellular release of mature IL-1ß and IL-18 during pyroptosis. In this chapter, we review our current understanding of GSDM proteins in physiological and pathological cell death, with more focused discussions on its structural basis for GSDM activation and pore formation.
Subject(s)
Neoplasm Proteins , Pyroptosis , Animals , Caspases/metabolism , Enzyme Activation , Humans , Inflammasomes , Intracellular Signaling Peptides and Proteins , Mice , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Phosphate-Binding Proteins , Structure-Activity RelationshipABSTRACT
The gasdermin (GSDM) family consists of gasdermin A (GSDMA), B (GSDMB), C (GSDMC), D (GSDMD), E or DNFA5 (GSDME), and DFNB59 in human. Expressed in the skin, gastrointestinal tract, and various immune cells, GSDMs mediate homeostasis and inflammation upon activation by caspases and unknown proteases. In particular, GSDMD is activated by inflammasome-activated caspases-1/-4/-5/-11 as well as a caspase-8-mediated pathway during Yersinia infection. These caspases cleave GSDMD to release its functional N-terminal fragment (GSDMD-NT) from its auto-inhibitory C-terminal fragment (GSDMD-CT). GSDMD-NTs bind to acid lipids in mammalian cell membranes and bacterial membranes, oligomerize, and insert into the membranes to form large transmembrane pores. Consequently, cellular contents including inflammatory cytokines are released and cells can undergo pyroptosis, a highly inflammatory form of cell death. In this chapter, we summarize recent research findings and present experimental procedures to obtain pure recombinant GSDMs for biochemical studies. We highlight a liposome-based assay that yields robust fluorescence signals for characterizing GSDM activities in vitro and may be applicable to other pore-forming proteins and ion channels in general.
Subject(s)
Biomarkers, Tumor/metabolism , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phosphate-Binding Proteins/metabolism , Receptors, Estrogen/metabolism , Animals , Biomarkers, Tumor/genetics , Caspases/genetics , Caspases/metabolism , DNA-Binding Proteins/genetics , Humans , Inflammasomes/metabolism , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Liposomes/metabolism , Neoplasm Proteins/genetics , Nerve Tissue Proteins/genetics , Phosphate-Binding Proteins/genetics , Pyroptosis/genetics , Pyroptosis/physiology , Receptors, Estrogen/geneticsABSTRACT
In the version of this article initially published, the label (CASP4-C285A-HA) above the second and fifth lanes in the right blot in Fig. 1e is incorrect; the correct label is CASP4-C258A-HA. Also, the two labels at right above the plot in Fig. 6c were switched; the far right label should be 'Co-housed Serpinb1a-/-' (in red font) and the label just to its left (above the fourth column) should be 'Co-housed WT' (in black font). Finally, the bottom two symbols in the key to Fig. 7d were switched; the red circle should identify 1CARD-SUMO (TEV) and the blue triangle should identify 1CARD-SUMO + SERPINB1 (TEV). The errors have been corrected in the HTML and PDF versions of the article.
ABSTRACT
Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation. Here we present cryo-EM and crystal structures of NLRP6 pyrin domain (PYD). We show that NLRP6 PYD alone is able to self-assemble into filamentous structures accompanied by large conformational changes and can recruit the ASC adaptor using PYD-PYD interactions. Using molecular dynamics simulations, we identify the surface that the NLRP6 PYD filament uses to recruit ASC PYD. We further find that full-length NLRP6 assembles in a concentration-dependent manner into wider filaments with a PYD core surrounded by the NBD and the LRR domain. These findings provide a structural understanding of inflammasome assembly by NLRP6 and other members of the NLR family.
Subject(s)
Inflammasomes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Amino Acid Sequence , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Immunity, Innate , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Protein MultimerizationABSTRACT
Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.