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DPP9 sequesters the C terminus of NLRP1 to repress inflammasome activation.

ABSTRACT: Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis^1-4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin^4-6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains^7-9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)^10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear^10,12-14. Here we report cryo-electron microscopy structures of the human NLRP1-DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1-DPP9 interaction and accelerates degradation of the N-terminal fragment¹⁰ to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome.

SUBMITTER: Hollingsworth LR

PROVIDER: S-EPMC8299537 | biostudies-literature |

REPOSITORIES: biostudies-literature

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NLRP1 variant M1184V decreases inflammasome activation in the context of DPP9 inhibition and asthma severity.

Project description:BackgroundNLRP1 is an innate immune sensor that can form cytoplasmic inflammasome complexes. Polymorphisms in NLRP1 are linked to asthma; however, there is currently no functional or mechanistic explanation for this.ObjectiveWe sought to clarify the role of NLRP1 in asthma pathogenesis.MethodsResults from the GALA II cohort study were used to identify a link between NLRP1 and asthma in Mexican Americans. In vitro and in vivo models for NLRP1 activation were applied to investigate the role of this inflammasome in asthma at the molecular level.ResultsWe document the association of an NLRP1 haplotype with asthma for which the single nucleotide polymorphism rs11651270 (M1184V) individually is the most significant. Surprisingly, M1184V increases NLRP1 activation in the context of N-terminal destabilization, but decreases NLRP1 activation on dipeptidyl peptidase 9 inhibition. In vitro studies demonstrate that M1184V increases binding to dipeptidyl peptidase 9, which can account for its inhibitory role in this context. In addition, in vivo data from a mouse model of airway inflammation reveal a protective role for NLRP1 inflammasome activation reducing eosinophilia in this setting.ConclusionsLinking our in vitro and in vivo results, we found that the NLRP1 variant M1184V reduces inflammasome activation in the context of dipeptidyl peptidase 9 inhibition and could thereby increase asthma severity. Our studies may have implications for the treatment of asthma in patients carrying this variant of NLRP1.

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Human NLRP1-DPP9 complex

Project description: Not available

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Project description: Not available

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Inflammasome sensor NLRP1 disease variant M1184V promotes autoproteolysis and DPP9 complex formation by stabilizing the FIIND domain.

Project description:The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the "function-to-find" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immunoprecipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity assays. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes.

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Human DPP9 represses NLRP1 inflammasome and protects against autoinflammatory diseases via both peptidase activity and FIIND domain binding.

Project description:The inflammasome is a critical molecular complex that activates interleukin-1 driven inflammation in response to pathogen- and danger-associated signals. Germline mutations in the inflammasome sensor NLRP1 cause Mendelian systemic autoimmunity and skin cancer susceptibility, but its endogenous regulation remains less understood. Here we use a proteomics screen to uncover dipeptidyl dipeptidase DPP9 as a novel interacting partner with human NLRP1 and a related inflammasome regulator, CARD8. DPP9 functions as an endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP8/9 inhibition via small molecule drugs and CRISPR/Cas9-mediated genetic deletion specifically activate the human NLRP1 inflammasome, leading to ASC speck formation, pyroptotic cell death, and secretion of cleaved interleukin-1β. Mechanistically, DPP9 interacts with a unique autoproteolytic domain (Function to Find Domain (FIIND)) found in NLRP1 and CARD8. This scaffolding function of DPP9 and its catalytic activity act synergistically to maintain NLRP1 in its inactive state and repress downstream inflammasome activation. We further identified a single patient-derived germline missense mutation in the NLRP1 FIIND domain that abrogates DPP9 binding, leading to inflammasome hyperactivation seen in the Mendelian autoinflammatory disease Autoinflammation with Arthritis and Dyskeratosis. These results unite recent findings on the regulation of murine Nlrp1b by Dpp8/9 and uncover a new regulatory mechanism for the NLRP1 inflammasome in primary human cells. Our results further suggest that DPP9 could be a multifunctional inflammasome regulator involved in human autoinflammatory diseases.

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Structural and biochemical mechanisms of NLRP1 inhibition by DPP9.

Project description:Nucleotide-binding domain, leucine-rich repeat receptors (NLRs) mediate innate immunity by forming inflammasomes. Activation of the NLR protein NLRP1 requires autocleavage within its function-to-find domain (FIIND)1-7. In resting cells, the dipeptidyl peptidases DPP8 and DPP9 interact with the FIIND of NLRP1 and suppress spontaneous NLRP1 activation8,9; however, the mechanisms through which this occurs remain unknown. Here we present structural and biochemical evidence that full-length rat NLRP1 (rNLRP1) and rat DPP9 (rDPP9) form a 2:1 complex that contains an autoinhibited rNLRP1 molecule and an active UPA-CARD fragment of rNLRP1. The ZU5 domain is required not only for autoinhibition of rNLRP1 but also for assembly of the 2:1 complex. Formation of the complex prevents UPA-mediated higher-order oligomerization of UPA-CARD fragments and strengthens ZU5-mediated NLRP1 autoinhibition. Structure-guided biochemical and functional assays show that both NLRP1 binding and enzymatic activity are required for DPP9 to suppress NLRP1 in human cells. Together, our data reveal the mechanism of DPP9-mediated inhibition of NLRP1 and shed light on the activation of the NLRP1 inflammasome.

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Project description:NLRP1 and CARD8 are related pattern-recognition receptors (PRRs) that detect intracellular danger signals and form inflammasomes. Both undergo autoproteolysis, generating N-terminal (NT) and C-terminal (CT) fragments. The proteasome-mediated degradation of the NT releases the CT from autoinhibition, but the stimuli that trigger NT degradation have not been fully elucidated. Here, we show that several distinct agents that interfere with protein folding, including aminopeptidase inhibitors, chaperone inhibitors, and inducers of the unfolded protein response, accelerate NT degradation. However, these agents alone do not trigger inflammasome formation because the released CT fragments are physically sequestered by the serine dipeptidase DPP9. We show that DPP9-binding ligands must also be present to disrupt these complexes and allow the CT fragments to oligomerize into inflammasomes. Overall, these results indicate that NLRP1 and CARD8 detect a specific perturbation that induces both protein folding stress and DPP9 ligand accumulation.

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NLRP1 inflammasome activation induces pyroptosis of hematopoietic progenitor cells.

Project description:Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1? but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1?-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.

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