scholarly journals NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain

2017 ◽  
Vol 214 (6) ◽  
pp. 1725-1736 ◽  
Author(s):  
Andrea Stutz ◽  
Carl-Christian Kolbe ◽  
Rainer Stahl ◽  
Gabor L. Horvath ◽  
Bernardo S. Franklin ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Cell Death ◽  
Proinflammatory Cytokines ◽  
Inflammasome Activation ◽  
Phosphorylation Sites ◽  
Danger Signals ◽  
Pyrin Domain ◽  
Charge Interaction ◽  
Phosphatase 2A ◽  
Recognition Receptor

NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge–charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.

scholarly journals Structural mechanism of CARD8 regulation by DPP9

2021 ◽  
Author(s):  
Humayun Sharif ◽  
L. Robert Hollingsworth ◽  
Andrew R. Griswold ◽  
Jeffrey C. Hsiao ◽  
Qinghui Wang ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Ternary Complex ◽  
Proteasomal Degradation ◽  
Full Length ◽  
Inflammasome Activation ◽  
Structural Mechanism ◽  
Danger Signals ◽  
Caspase 1 ◽  
Recognition Receptor

SUMMARYCARD8 is a germline-encoded pattern recognition receptor that detects intracellular danger signals. Like the related inflammasome sensor NLRP1, CARD8 undergoes constitutive autoprocessing within its function-to-find domain (FIIND), generating two polypeptides that stay associated and autoinhibited. Certain pathogen- and danger-associated activities, including the inhibition of the serine dipeptidases DPP8 and DPP9 (DPP8/9), induce the proteasome-mediated degradation of the N-terminal (NT) fragment, releasing the C-terminal (CT) fragment to form a caspase-1 activating inflammasome. DPP8/9 also bind directly to the CARD8 FIIND, but the role that this interaction plays in CARD8 inflammasome regulation is not yet understood. Here, we solved several cryo-EM structures of CARD8 bound to DPP9, with or without the DPP inhibitor Val-boroPro (VbP), which revealed a ternary complex composed of one DPP9, the full-length CARD8, and one CARD8-CT. Through structure-guided biochemical and cellular experiments, we demonstrated that DPP9’s structure restrains CARD8-CT after proteasomal degradation. Moreover, although DPP inhibitors do not directly displace CARD8 from DPP9 in vitro, we show that they can nevertheless destabilize this complex in cells. Overall, these results demonstrate that DPP8/9 inhibitors cause CARD8 inflammasome activation via at least two distinct mechanisms, one upstream and one downstream of the proteasome.

scholarly journals Structure, Activation and Regulation of NLRP3 and AIM2 Inflammasomes

2021 ◽  
Vol 22 (2) ◽  
pp. 872
Author(s):  
Meenakshi Sharma ◽  
Eva de Alba
Keyword(s):  
Cell Death ◽  
Immune Responses ◽  
Proinflammatory Cytokines ◽  
Interaction Mechanism ◽  
Inflammasome Activation ◽  
Danger Signals ◽  
Effector Caspase ◽  
Cardiometabolic Disorders ◽  
Recent Advancement ◽  
Signaling Process

The inflammasome is a three-component (sensor, adaptor, and effector) filamentous signaling platform that shields from multiple pathogenic infections by stimulating the proteolytical maturation of proinflammatory cytokines and pyroptotic cell death. The signaling process initiates with the detection of endogenous and/or external danger signals by specific sensors, followed by the nucleation and polymerization from sensor to downstream adaptor and then to the effector, caspase-1. Aberrant activation of inflammasomes promotes autoinflammatory diseases, cancer, neurodegeneration, and cardiometabolic disorders. Therefore, an equitable level of regulation is required to maintain the equilibrium between inflammasome activation and inhibition. Recent advancement in the structural and mechanistic understanding of inflammasome assembly potentiates the emergence of novel therapeutics against inflammasome-regulated diseases. In this review, we have comprehensively discussed the recent and updated insights into the structure of inflammasome components, their activation, interaction, mechanism of regulation, and finally, the formation of densely packed filamentous inflammasome complex that exists as micron-sized punctum in the cells and mediates the immune responses.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

scholarly journals Inflammation in nonischemic heart disease: initiation by cardiomyocyte CaMKII and NLRP3 inflammasome signaling

2019 ◽  
Vol 317 (5) ◽  
pp. H877-H890 ◽  
Author(s):  
Takeshi Suetomi ◽  
Shigeki Miyamoto ◽  
Joan Heller Brown
Keyword(s):  
Cell Death ◽  
Heart Disease ◽  
Nlrp3 Inflammasome ◽  
Immune Cell ◽  
Cell Damage ◽  
Nuclear Factor Κb ◽  
Inflammasome Activation ◽  
Monocyte Chemoattractant Protein 1 ◽  
Pyrin Domain ◽  
Cytokines And Chemokines

There is substantial evidence that chronic heart failure in humans and in animal models is associated with inflammation. Ischemic interventions such as myocardial infarction lead to necrotic cell death and release of damage associated molecular patterns, factors that signal cell damage and induce expression of proinflammatory chemokines and cytokines. It has recently become evident that nonischemic interventions are also associated with increases in inflammatory genes and immune cell accumulation in the heart and that these contribute to fibrosis and ventricular dysfunction. How proinflammatory responses are elicited in nonischemic heart disease which is not, at least initially, associated with cell death is a critical unanswered question. In this review we provide evidence supporting the hypothesis that cardiomyocytes are an initiating site of inflammatory gene expression in response to nonischemic stress. Furthermore we discuss the role of the multifunctional Ca2+/calmodulin-regulated kinase, CaMKIIδ, as a transducer of stress signals to nuclear factor-κB activation, expression of proinflammatory cytokines and chemokines, and priming and activation of the NOD-like pyrin domain-containing protein 3 (NLRP3) inflammasome in cardiomyocytes. We summarize recent evidence that subsequent macrophage recruitment, fibrosis and contractile dysfunction induced by angiotensin II infusion or transverse aortic constriction are ameliorated by blockade of CaMKII, of monocyte chemoattractant protein-1/C-C chemokine receptor type 2 signaling, or of NLRP3 inflammasome activation.

Inhibitory pattern recognition receptors

2021 ◽  
Vol 219 (1) ◽  
Author(s):  
Matevž Rumpret ◽  
Helen J. von Richthofen ◽  
Victor Peperzak ◽  
Linde Meyaard
Keyword(s):  
Pattern Recognition ◽  
Cell Death ◽  
Immune System ◽  
Immune Responses ◽  
Pattern Recognition Receptors ◽  
Inhibitory Receptors ◽  
Danger Signals ◽  
Molecular Pattern ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

Pathogen- and damage-associated molecular patterns are sensed by the immune system’s pattern recognition receptors (PRRs) upon contact with a microbe or damaged tissue. In situations such as contact with commensals or during physiological cell death, the immune system should not respond to these patterns. Hence, immune responses need to be context dependent, but it is not clear how context for molecular pattern recognition is provided. We discuss inhibitory receptors as potential counterparts to activating pattern recognition receptors. We propose a group of inhibitory pattern recognition receptors (iPRRs) that recognize endogenous and microbial patterns associated with danger, homeostasis, or both. We propose that recognition of molecular patterns by iPRRs provides context, helps mediate tolerance to microbes, and helps balance responses to danger signals.

scholarly journals Novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells

2014 ◽  
Vol 45 (2) ◽  
pp. 428-439 ◽  
Author(s):  
Aline Schögler ◽  
Brigitte S. Kopf ◽  
Michael R. Edwards ◽  
Sebastian L. Johnston ◽  
Carmen Casaulta ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pattern Recognition ◽  
Cell Death ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
Pattern Recognition Receptor ◽  
Bronchial Epithelial ◽  
Pulmonary Exacerbations ◽  
Pre Treatment ◽  
Recognition Receptor

Virus-associated pulmonary exacerbations, often associated with rhinoviruses (RVs), contribute to cystic fibrosis (CF) morbidity. Currently, there are only a few therapeutic options to treat virus-induced CF pulmonary exacerbations. The macrolide antibiotic azithromycin has antiviral properties in human bronchial epithelial cells. We investigated the potential of azithromycin to induce antiviral mechanisms in CF bronchial epithelial cells.Primary bronchial epithelial cells from CF and control children were infected with RV after azithromycin pre-treatment. Viral RNA, interferon (IFN), IFN-stimulated gene and pattern recognition receptor expression were measured by real-time quantitative PCR. Live virus shedding was assessed by assaying the 50% tissue culture infective dose. Pro-inflammatory cytokine and IFN-β production were evaluated by ELISA. Cell death was investigated by flow cytometry.RV replication was increased in CF compared with control cells. Azithromycin reduced RV replication seven-fold in CF cells without inducing cell death. Furthermore, azithromycin increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels. While stimulating antiviral responses, azithromycin did not prevent virus-induced pro-inflammatory responses.Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway. Clinical studies are needed to elucidate the potential of azithromycin in the management and prevention of RV-induced CF pulmonary exacerbations.

scholarly journals Human caspase-1 autoproteolysis is required for ASC-dependent and -independent inflammasome activation

2019 ◽  
Author(s):  
Daniel P. Ball ◽  
Cornelius Y. Taabazuing ◽  
Andrew R. Griswold ◽  
Elizabeth L. Orth ◽  
Sahana D. Rao ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Cell Death ◽  
Pattern Recognition Receptors ◽  
Inflammasome Activation ◽  
Adapter Protein ◽  
Large Structure ◽  
Catalytic Subunits ◽  
Caspase 1 ◽  
Human Caspase

AbstractPathogen-related signals induce a number of cytosolic pattern-recognition receptors (PRRs) to form canonical inflammasomes, which activate pro-caspase-1 and trigger pyroptotic cell death. All well-studied PRRs oligomerize with the pro-caspase-1-adapter protein ASC to generate a single large structure in the cytosol, which induces the autoproteolysis and activation of the pro-caspase-1 zymogen. However, several PRRs can also directly interact with pro-caspase-1 without ASC, forming much smaller “ASC-independent” inflammasomes. It is currently thought that pro-caspase-1 autoproteolysis does not occur during, and is not required for, ASC-independent inflammasome activation. Here, we show that the related human PRRs NLRP1 and CARD8 exclusively form ASC-dependent and ASC-independent inflammasomes, respectively, identifying CARD8 as the first PRR that cannot form an ASC-containing signaling platform. Despite their different structures, we discovered that both the NLRP1 and CARD8 inflammasomes require pro-caspase-1 autoproteolysis between the small and large catalytic subunits to induce pyroptosis. Thus, pro-caspase-1 self-cleavage is an obligate regulatory step in the activation of human canonical inflammasomes.

scholarly journals Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation

2021 ◽  
Author(s):  
Inga V. Hochheiser ◽  
Heide Behrmann ◽  
Gregor Hagelueken ◽  
Juan F. Rodriguez-Alcazar ◽  
Anja Kopp ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Responses ◽  
Molecular Model ◽  
Adaptor Protein ◽  
Continuous Transition ◽  
Danger Signals ◽  
Caspase 1 ◽  
Pyrin Domain ◽  
Protein Elongation ◽  
Family Cytokine

Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming Nod-like receptors with the adaptor protein ASC mediate oligomerization into helical filamentous assemblies. These supramolecular organizing centers recruit and activate caspase-1, which results in IL-1β family cytokine maturation and pyroptotic cell death. The molecular details of the critical step in signal transduction of inflammasome signaling, however, remain ill-defined. Here, we describe the cryo-EM structure of the human NLRP3 PYD filament at 3.6 Ang resolution. We identify a unique pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends that we designate as A- and B-end, respectively. Coupling a titration polymerization assay to cryo-EM, we demonstrate that the ASC adaptor protein elongation on NLRP3 PYD filament seeds is unidirectional, associating exclusively to the B-end of the NLRP3 filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing for a continuous transition between NLRP3 as the nucleation seed and ASC as the elongator. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3-NEK7, ASC, and Caspase-1 proteins.

scholarly journals Embryonic stem cell-derived exosomes inhibit doxorubicin-induced TLR4-NLRP3-mediated cell death-pyroptosis

2019 ◽  
Vol 317 (2) ◽  
pp. H460-H471 ◽  
Author(s):  
Zahra Tavakoli Dargani ◽  
Dinender K. Singla
Keyword(s):  
Cell Death ◽  
Proinflammatory Cytokines ◽  
Embryonic Stem ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Caspase 1 ◽  
Pyrin Domain ◽  
Tnf Α

Doxorubicin (Dox)-induced cardiac side effects are regulated through increased oxidative stress and apoptosis. However, it remains unknown whether Dox induces the specific inflammatory-mediated form of cell death called pyroptosis. The current study is undertaken to determine whether Dox induces pyroptosis in an in vitro model and to test the potential of exosomes derived from embryonic stem cells (ES-Exos) in inhibiting pyroptosis. H9c2 cells were exposed to Dox to generate pyroptosis and then subsequently treated with exosomes to investigate the protective effects of ES-Exos. Mouse embryonic fibroblast-exosomes (MEF-Exos) were used as a cell line control. We confirmed pyroptosis by analyzing the presence of Toll-like receptor 4 (TLR4)-pyrin domain containing-3 (NLRP3) inflammasome that initiates pyroptosis, which was further confirmed with pyroptotic markers caspase-1, IL-1β, caspase-11, and gasdermin-D. The presence of inflammation was confirmed for proinflammatory cytokines, TNF-α, and IL-6. Our data show that Dox exposure significantly ( P < 0.05) increases expression of TLR4, NLRP3, pyroptotic markers (caspase-1, IL-1β, caspase-11, and gasdermin-D), and proinflammatory cytokines (TNF-α and IL-6) in H9c2 cells. The increased expression of inflammasome, pyroptosis, and inflammation was significantly ( P < 0.05) inhibited by ES-Exos. Interestingly, our cell line control, MEF-Exos, did not show any protective effects. Furthermore, our cytokine array data suggest increased anti-inflammatory (IL-4, IL-9, and IL-13) and decreased proinflammatory cytokines (Fas ligand, IL-12, and TNF-α) in ES-Exos, suggesting that anti-inflammatory cytokines might be mediating the protective effects of ES-Exos. In conclusion, our data show that Dox induces pyroptotic cell death in the H9c2 cell culture model and is attenuated via treatment with ES-Exos. NEW & NOTEWORTHY Doxorubicin (Dox)-induced cardiotoxicity is mediated through increased oxidative stress, apoptosis, and necrosis. We report for the first time as per the best of our knowledge that Dox initiates Toll-like receptor 4 and pyrin domain containing-3 inflammasome formation and induces caspase-1-mediated inflammatory pyroptotic cell death in H9c2 cells. Moreover, we establish that inflammation and pyroptosis is inhibited by embryonic stem cell-derived exosomes that could be used as a future therapeutic option to treat Dox-induced cardiotoxicity.

scholarly journals Lytic Cell Death Mechanisms in Human Respiratory Syncytial Virus-Infected Macrophages: Roles of Pyroptosis and Necroptosis

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 932
Author(s):  
Lori Bedient ◽  
Swechha Mainali Pokharel ◽  
Kim R. Chiok ◽  
Indira Mohanty ◽  
Sierra S. Beach ◽  
...  
Keyword(s):  
Cell Death ◽  
Respiratory Syncytial Virus ◽  
Kinase Domain ◽  
Human Respiratory Syncytial Virus ◽  
Inflammasome Activation ◽  
Pyrin Domain ◽  
Cellular Debris ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Cell Death Mechanisms

Human respiratory syncytial virus (RSV) is the most common cause of viral bronchiolitis and pneumonia in infants and children worldwide. Inflammation induced by RSV infection is responsible for its hallmark manifestation of bronchiolitis and pneumonia. The cellular debris created through lytic cell death of infected cells is a potent initiator of this inflammation. Macrophages are known to play a pivotal role in the early innate immune and inflammatory response to viral pathogens. However, the lytic cell death mechanisms associated with RSV infection in macrophages remains unknown. Two distinct mechanisms involved in lytic cell death are pyroptosis and necroptosis. Our studies revealed that RSV induces lytic cell death in macrophages via both of these mechanisms, specifically through the ASC (Apoptosis-associated speck like protein containing a caspase recruitment domain)-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome activation of both caspase-1 dependent pyroptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), as well as a mixed lineage kinase domain like pseudokinase (MLKL)-dependent necroptosis. In addition, we demonstrated an important role of reactive oxygen species (ROS) during lytic cell death of RSV-infected macrophages.

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