RESUMO
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.
Assuntos
Toxinas Bacterianas/metabolismo , Enterococcus , Leucócitos Mononucleares , Fatores de Virulência/metabolismo , Animais , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Cavalos , Camundongos , Testes de Sensibilidade Microbiana , SuínosRESUMO
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.
Assuntos
Alarminas/metabolismo , Endotoxemia/imunologia , Galectina 1/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alarminas/deficiência , Alarminas/genética , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Endotoxemia/patologia , Feminino , Galectina 1/sangue , Galectina 1/deficiência , Galectina 1/genética , Células HeLa , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos Comuns de Leucócito/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Necroptose , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Células RAW 264.7 , Sepse/sangue , Sepse/diagnóstico , Transdução de Sinais , Regulação para CimaRESUMO
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.
Assuntos
Dissulfiram/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sepse/tratamento farmacológico , Animais , Caspase 1/genética , Caspase 1/metabolismo , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Linhagem Celular Tumoral , Dissulfiram/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Piroptose/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sepse/imunologia , Células Sf9 , SpodopteraRESUMO
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.
RESUMO
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.
Assuntos
Caspases/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Serpinas/imunologia , Animais , Caspases/genética , Caspases/metabolismo , Linhagem Celular , Células Cultivadas , Ativação Enzimática/imunologia , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Piroptose/efeitos dos fármacos , Piroptose/imunologia , Células RAW 264.7 , Interferência de RNA , Serina Proteases/imunologia , Serina Proteases/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células THP-1 , Células U937RESUMO
Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1. We found that PYD and CARD both form filaments. Activated AIM2 and NLRP3 nucleate PYD filaments of ASC, which, in turn, cluster the CARD of ASC. ASC thus nucleates CARD filaments of caspase-1, leading to proximity-induced activation. Endogenous NLRP3 inflammasome is also filamentous. The cryoelectron microscopy structure of ASC(PYD) filament at near-atomic resolution provides a template for homo- and hetero-PYD/PYD associations, as confirmed by structure-guided mutagenesis. We propose that ASC-dependent inflammasomes in both families share a unified assembly mechanism that involves two successive steps of nucleation-induced polymerization. PAPERFLICK:
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Inflamassomos/química , Sequência de Aminoácidos , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Humanos , Inflamassomos/metabolismo , Inflamassomos/ultraestrutura , Interleucina-1beta/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas Nucleares/metabolismo , Polimerização , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Proteínas de Bactérias , Gasderminas , Proteínas Citotóxicas Formadoras de Poros , Animais , Humanos , Camundongos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Sequência Conservada , Microscopia Crioeletrônica , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Citotóxicas Formadoras de Poros/ultraestrutura , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Piroptose , Shigella , Especificidade da Espécie , Gasderminas/química , Gasderminas/metabolismo , Gasderminas/ultraestruturaRESUMO
The interleukin-1 (IL-1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL-1 while retaining viability, yet it is unclear how IL-1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL-1ß secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1ß transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation.
Assuntos
Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Fosfato , Transporte Proteico/fisiologia , Piroptose/imunologiaRESUMO
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.
Assuntos
Inflamassomos/química , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Proteínas de Ligação a Fosfato/química , Animais , Caspase 1/metabolismo , Microscopia Crioeletrônica , Humanos , Interleucina-1/metabolismo , Camundongos Endogâmicos C57BL , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
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.
Assuntos
Anfioxos , Piroptose , Animais , Piroptose/fisiologia , Anfioxos/metabolismo , Gasderminas , Proteínas de Neoplasias/metabolismo , Mecanismos de DefesaRESUMO
Tumor necrosis factor (TNF) can drive inflammation, cell survival, and death. While ubiquitylation-, phosphorylation-, and nuclear factor κB (NF-κB)-dependent checkpoints suppress the cytotoxic potential of TNF, it remains unclear whether ubiquitylation can directly repress TNF-induced death. Here, we show that ubiquitylation regulates RIPK1's cytotoxic potential not only via activation of downstream kinases and NF-kB transcriptional responses, but also by directly repressing RIPK1 kinase activity via ubiquitin-dependent inactivation. We find that the ubiquitin-associated (UBA) domain of cellular inhibitor of apoptosis (cIAP)1 is required for optimal ubiquitin-lysine occupancy and K48 ubiquitylation of RIPK1. Independently of IKK and MK2, cIAP1-mediated and UBA-assisted ubiquitylation suppresses RIPK1 kinase auto-activation and, in addition, marks it for proteasomal degradation. In the absence of a functional UBA domain of cIAP1, more active RIPK1 kinase accumulates in response to TNF, causing RIPK1 kinase-mediated cell death and systemic inflammatory response syndrome. These results reveal a direct role for cIAP-mediated ubiquitylation in controlling RIPK1 kinase activity and preventing TNF-mediated cytotoxicity.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/fisiologia , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose , Células HEK293 , Humanos , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , UbiquitinaçãoRESUMO
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.
Assuntos
Antígenos CD18 , Neutrófilos , Animais , Antígenos CD18/metabolismo , Cálcio/metabolismo , Adesão Celular , Guanosina , Guanosina Trifosfato/metabolismo , Humanos , Interleucina-8/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Polifosfatos , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel ß-barrel is formed by two ß-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning ß-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer.
Assuntos
Microscopia Crioeletrônica , Proteínas/química , Proteínas/ultraestrutura , Animais , Membrana Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipídeos de Membrana/metabolismo , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/ultraestrutura , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/ultraestrutura , Neoplasias/genética , Perforina/química , Perforina/metabolismo , Proteínas de Ligação a Fosfato , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Relação Estrutura-AtividadeRESUMO
Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1ß. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Inflamassomos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Piroptose , Sequência de Aminoácidos , Animais , Cardiolipinas/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sequência Conservada/genética , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipossomos/química , Lipossomos/metabolismo , Listeria monocytogenes/citologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/farmacologia , Proteínas de Ligação a Fosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Porosidade/efeitos dos fármacos , Multimerização Proteica/genética , Estrutura Terciária de Proteína/genética , Transporte Proteico , Piroptose/efeitos dos fármacos , Piroptose/genética , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismoRESUMO
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.
Assuntos
Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica , Multimerização ProteicaRESUMO
Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD-CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/química , Proteínas de Ligação ao Cálcio/química , Caspase 1/química , Microscopia Crioeletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Ativação Enzimática , Humanos , Inflamassomos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismoRESUMO
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.
Assuntos
Proteínas de Neoplasias , Piroptose , Animais , Caspases/metabolismo , Ativação Enzimática , Humanos , Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a Fosfato , Relação Estrutura-AtividadeRESUMO
The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.
Assuntos
Acetiltransferases/química , Acetiltransferases/metabolismo , Regulação da Expressão Gênica , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Acetilação , Acetiltransferases/genética , Sequência de Aminoácidos , Western Blotting , Imunoprecipitação da Cromatina , Histona Acetiltransferases/genética , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transativadores/genéticaRESUMO
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.
Assuntos
Inflamassomos , Proteínas NLR , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Proteínas NLR/metabolismo , Animais , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/metabolismo , Transdução de Sinais/imunologia , Imunidade Inata , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
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.