ASC-CARD:Caspase-1-CARD octamer
Ontology highlight
ABSTRACT:
SUBMITTER: Hao Wu
PROVIDER: EMPIAR-10566 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes.
Robert Hollingsworth L L David Liron L Li Yang Y Griswold Andrew R AR Ruan Jianbin J Sharif Humayun H Fontana Pietro P Orth-He Elizabeth L EL Fu Tian-Min TM Bachovchin Daniel A DA Wu Hao H
Nature communications 20210108 1
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-ass ...[more]
Similar Datasets
Project description: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.
| S-EPMC6205419 | biostudies-literature
Project description:The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display.
| S-EPMC3247988 | biostudies-literature
Project description:Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS and induce pyroptosis. Caspase-11 contains two components, an N-terminal caspase recruitment domain (CARD) and a C-terminal catalytic domain. The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11. However, the activation mechanism of caspase-11 remains unclear. In this study, we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution. Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1-2 helix mediating homotypic CARD interactions. Importantly, mutations of the helix H1-2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells. Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model. This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition, leading to the caspase-11 activation.
| S-EPMC7552397 | biostudies-literature
Project description:Direct recruitment and activation of caspase-9 by Apaf-1 through the homophilic CARD/CARD (Caspase Recruitment Domain) interaction is critical for the activation of caspases downstream of mitochondrial damage in apoptosis. Here we report the solution structure of the Apaf-1 CARD domain and its surface of interaction with caspase-9 CARD. Apaf-1 CARD consists of six tightly packed amphipathic alpha-helices and is topologically similar to the RAIDD CARD, with the exception of a kink observed in the middle of the N-terminal helix. By using chemical shift perturbation data, the homophilic interaction was mapped to the acidic surface of Apaf-1 CARD centered around helices 2 and 3. Interestingly, a significant portion of the chemically perturbed residues are hydrophobic, indicating that in addition to the electrostatic interactions predicted previously, hydrophobic interaction is also an important driving force underlying the CARD/CARD interaction. On the basis of the identified functional residues of Apaf-1 CARD and the surface charge complementarity, we propose a model of CARD/CARD interaction between Apaf-1 and caspase-9.
| S-EPMC18022 | biostudies-literature
Project description:The ASC (apoptosis speck-like protein) is a key component of multimeric protein complexes that mediate inflammation and host defence. Comprising a PYD (Pyrin) domain and a CARD (caspase activation and recruitment domain), ASC functions downstream of NLRs (nucleotide-binding domain, leucine-rich repeat-containing receptors) and AIM2 (absent in melanoma 2) through the formation of supramolecular structures termed inflammasomes. However, the mechanism underlying ASC signalling and its dependency on oligomeric arrangements in inflammasome formation remain poorly understood. When expressed in cells, ASC forms discrete foci (called 'specks') typically with one speck per cell. We employed a BiFC (bimolecular fluorescence complementation) system to investigate and visualize ASC foci formation in living cells. We demonstrated that the CARD of ASC plays a central role in ASC inflammasome assembly, representing the minimal unit capable of forming foci in conjunction with the caspase 1 CARD. Mutational studies point to multiple surfaces on the ASC CARD and two predominant areas on the caspase 1 CARD mediating the formation of ASC/caspase 1 foci. The lack of foci formation for ASC CARD mutants correlates with a loss of IL-1β (interleukin 1β) processing in response to NLRP (NLR family, PYD domain-containing) 3 or AIM2 agonists in RAW264.7 cell reconstitution assays. Analogously, we show that productive formation of the Salmonella typhimurium-induced NLRC4 (NLR family CARD domain-containing protein 4) inflammasome is dependent on ASC-CARD-mediated platform formation. Thus the results of the present study depict a central role of CARDs in the formation of ASC signalling platforms and provide an important tool for investigation of CARD-dependent networks.
| S-EPMC3966062 | biostudies-literature
Project description:Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative bacteria and are pathogen-associated molecular patterns recognized by the TLR4/MD2 complex that induces an inflammatory response. Recently, the cytosolic receptors caspase-4/-5/-11 that bind LPS inside the cell and trigger inflammasome activation or pyroptosis, have been identified. Despite the important roles of caspase-4 in human immune responses, few studies have investigated its biochemical characteristics and interactions with LPS. Since caspase-4 (C258A) purified from an Escherichia coli host forms aggregates, monomeric proteins including full-length caspase-4, caspase-4 (C258A), and the CARD domain of caspase-4 have been purified from the insect cell system. Here, we report the overexpression and purification of monomeric caspase-4 (C258A) and CARD domain from E. coli and demonstrate that purified caspase-4 (C258A) and CARD domain bind large LPS micelles and disaggregate them to small complexes. As the molar ratio of caspase-4 to LPS increases, the size of the caspase-4/LPS complex decreases. Our results present a new function of caspase-4 and set the stage for structural and biochemical studies, and drug discovery targeting LPS/caspase-4 interactions by establishing a facile purification method to obtain large quantities of purified caspase-4 (C258A) and the CARD domain.
| S-EPMC6351570 | biostudies-literature
Project description:The human protein ASC is a key mediator in apoptosis and inflammation. Through its two death domains (pyrin and CARD) ASC interacts with cell death executioners, acts as an essential adapter for inflammasome integrity, and oligomerizes into functional supramolecular assemblies. However, these functions are not understood at the structural-dynamic level. This study reports the solution structure and interdomain dynamics of full-length ASC. The pyrin and CARD domains are structurally independent six-helix bundle motifs connected by a 23-residue linker. The CARD structure reveals two distinctive characteristics; helix 1 is not fragmented as in all other known CARDs, and its electrostatic surface shows a uniform distribution of positive and negative charges, whereas these are commonly separated into two areas in other death domains. The linker adopts residual structure resulting in a back-to-back orientation of the domains, which avoids steric interference of each domain with the binding site of the other. NMR relaxation experiments show that the linker is flexible despite the residual structure. This flexibility could help expand the relative volume occupied by each domain, thus increasing the capture radius for effectors. Based on the ASC structure, a tentative model is proposed to illustrate how ASC oligomerizes via CARD and pyrin homophilic interactions. Moreover, ASC oligomers have been analyzed by atomic force microscopy, showing a predominant species of disk-like particles of approximately 12-nm diameter and approximately 1-nm height. Taken together, these results provide structural insight into the behavior of ASC as an adapter molecule.
| S-EPMC2781708 | biostudies-literature
Project description:The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-? levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.
| S-EPMC3438500 | biostudies-other
Project description:Legionella pneumophila is a Gram-negative, flagellated bacterium that survives in phagocytes and causes Legionnaires' disease. Upon infection of mammalian macrophages, cytosolic flagellin triggers the activation of Naip/NLRC4 inflammasome, which culminates in pyroptosis and restriction of bacterial replication. Although NLRC4 and caspase-1 participate in the same inflammasome, Nlrc4-/- mice and their macrophages are more permissive to L. pneumophila replication compared with Casp1/11-/-. This feature supports the existence of a pathway that is NLRC4-dependent and caspase-1/11-independent. Here, we demonstrate that caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in response to flagellin-positive bacteria. Accordingly, caspase-8 is activated in Casp1/11-/- macrophages in a process dependent on flagellin, Naip5, NLRC4 and ASC. Silencing caspase-8 in Casp1/11-/- cells culminated in macrophages that were as susceptible as Nlrc4-/- for the restriction of L. pneumophila replication. Accordingly, macrophages and mice deficient in Asc/Casp1/11-/- were more susceptible than Casp1/11-/- and as susceptible as Nlrc4-/- for the restriction of infection. Mechanistically, we found that caspase-8 activation triggers gasdermin-D-independent pore formation and cell death. Interestingly, caspase-8 is recruited to the Naip5/NLRC4/ASC inflammasome in wild-type macrophages, but it is only activated when caspase-1 or gasdermin-D is inhibited. Our data suggest that caspase-8 activation in the Naip5/NLRC4/ASC inflammasome enable induction of cell death when caspase-1 or gasdermin-D is suppressed.
| S-EPMC5542441 | biostudies-literature
Project description:Apaf-1 facilitates the proteolytic activation of procaspase-9 and maintains the hyperactive state of the processed caspase-9. The underlying molecular mechanisms for these activities remain poorly characterized. Here we report that the isolated Apaf-1 caspase recruitment domain (CARD) forms a large hetero-oligomer with the active caspase-9. The catalytic activity of caspase-9 is significantly enhanced in this complex, demonstrating that Apaf-1 CARD allosterically up-regulates caspase-9 activity. Point mutations that inactivate the interactions between Apaf-1 CARD and the prodomain of caspase-9 also abolished the formation of this complex. Based on these observations, we discuss the implications of this complex on the observed Apaf-1 function.
| S-EPMC123625 | biostudies-literature