Novel mode of filament formation in UPA-promoted CARD8 and NLRP1 Inflammasomes

Abstract

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 subdomain of a function-to-find domain (FIIND) for self-oligomerization and recruitment of the inflammasome adaptor ASC and/or caspase-1. 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. We discover that subunits in the central NLRP1 CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. The thick NLRP1 filament only forms with the presence of UPA, which we hypothesize drives the intrinsic propensity for NLRP1 CARD dimerization. Structural analyses provide insights on the requirement of ASC for NLRP1-CT signalling and the contrasting direct recruitment of caspase-1 by CARD8-CT.Additionally, we present a low-resolution 4 ASC CARD -4 caspase-1 CARD octamer structure, illustrating that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. These structures capture the architecture and specificity of CARD inflammasome polymerization in NLRP1 and CARD8.Innate immune pathways recognize and respond to a diverse array of intracellular threats. In one such pathway, cells identify and amplify danger signals through supramolecular signalling complexes called canonical inflammasomes 1,2 . Upon recognition of intracellular molecular patterns indicative of pathogens or endogenous damage, sensor proteins facilitate inflammasome assembly by undergoing large conformational changes that lead to their oligomerization 3,4 .Nucleotide binding domains (NBDs) often drive this self-oligomerization to cluster death-fold domains 5,6 , which in turn recruit downstream adapter and effector molecules. These death-fold domains, including pyrin domains (PYD) and caspase recruitment domains (CARD), participate in homotypic (CARD-CARD or PYD-PYD) interactions that ultimately lead to polymerization of caspase-1 into filaments 3,4,7-9 . This process increases the local concentration of caspase catalytic domains to facilitate its homo-dimerization and autoproteolysis, resulting in its activation 1-3 . Active caspase-1, a cysteine protease, then processes pro-inflammatory cytokines, including pro-IL-1β and pro-IL-18, to their bioactive forms and cleaves the pore-forming protein gasdermin-D (GSDMD) to promote cytokine release and often concomitant lytic cell death termed pyroptosis 10-12 .Two unique sensor proteins, NLRP1 and CARD8, mediate inflammasome formation through their CARD-containing C-terminal fragment (CT) generated upon functional degradation of their respective N-terminal fragment (NT) by the proteasome 13,14 . This unusual mechanism of infl...

Structural mechanism of CARD8 regulation by DPP9

“…Despite maintaining FIIND autoprocessing, interface III mutations completely abolished inflammasome activity, even in the presence of the activating ligand VbP (Figure 3D). We reasoned that the observed UPA-UPA interface might be preserved on the inflammasome filament, as the UPA itself promotes CARD oligomerization and inflammasome activity (Hollingsworth et al, 2020a; Qin et al, 2020). Additionally, NLRP1 interface III mutations abolished inflammasome signaling in cells (Hollingsworth et al, 2020b) and disrupted filament formation in vitro (Huang et al, 2020).…”

“…However, it should be noted that our studies suggest that CARD8 and NLRP1 may have also both evolved to sense a single endogenous danger signal triggered by DPP8/9 inhibition. Another important difference between CARD8 and NLRP1 is the structure of assembled inflammasome: while CARD8 can only directly recruit caspase-1 and activate rapid pyroptosis without causing cytokine maturation, NLRP1 recruits caspase-1 via ASC to elicit an inflammatory signaling cascade with cytokine secretion and pyroptotic cell death (Ball et al, 2020; Hollingsworth et al, 2020a; Qin et al, 2020). In this context, it is possible that NLRP1 activation is more pro-inflammatory, as exemplified by the association of NLRP1 hyperactivation with a series of severe skin pro-inflammatory diseases (Grandemange et al, 2017; Jin et al, 2007; Levandowski et al, 2013; Zhong et al, 2016; Zhong et al, 2018).…”

“…The cryo-EM maps were first fit with the crystal structure of DPP9 dimer (PDB ID: 6EOQ) (Ross et al, 2018). A homology model of CARD8-FIIND was generated with Schrodinger Prime (Jacobson et al, 2002) using the structure of NLRP1-FIIND (Hollingsworth et al, 2020a; Qin et al, 2020) as template. Manual adjustment and de novo building of missing segments, rigid-body fitting, flexible fitting, and segment-based real-space refinement were performed in distinct parts of the initial model to fit in the density in Coot (Emsley et al, 2010), with help of UCSF-Chimera (Goddard et al, 2007) and real-space refinement in Phenix (Klaholz, 2019).…”

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

“…We hypothesize that the same UPA-UPA interaction surface as observed in the 2:1 NLRP1-DPP9 complex is utilized in the higher order UPA-CARD filament. Indeed, negative staining EM showed that the wild-type UPA-CARD of hNLRP1 formed filamentous structures 25,26 (Fig. 5b), but not the UPA-UPA interface mutants, UPA-CARD P1278E and UPA-CARD L1281E (Fig.…”

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

“…We hypothesize that the same UPA-UPA interaction surface as observed in the 2:1 NLRP1-DPP9 complex is utilized in the higher order UPA-CARD filament. Indeed, negative staining EM showed that the wild-type UPA-CARD of hNLRP1 formed filamentous structures 25,26 (Fig. 5b), but not the UPA-UPA interface mutants, UPA-CARD P1278E and UPA-CARD L1281E (Fig.…”