Mechanism 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 (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. 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. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD–caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

The NLRP1 Inflammasome Induces Pyroptosis in Human Corneal Epithelial Cells

“… 38 Second, the CARD8 inflammasome, unlike the NLRP1 inflammasome, does not efficiently process pro-IL-1β and 18. 36 , 45 Third, the long double stranded RNA mimetic poly(I:C) was recently reported to activate NLRP1, but not CARD8. 46 Fourth, a small molecule we recently identified called CQ31 selectively activates the CARD8 inflammasome without simultaneously activating the NLRP1 inflammasome (Rao SD, Chen Q, Wang Q, et al M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome.…”

“…S1 B). 28 , 29 , 36 Potent DPP8/9 inhibitors, including Val-boroPro (VbP), activate NLRP1 via both mechanisms. 26 , 31 , 37 …”

P38 kinases mediate NLRP1 inflammasome activation after ribotoxic stress response and virus infection

“…Human NLRP1 exhibits a unique domain structure and contains both an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD) (Martinon et al, 2002; Yu et al, 2017). Autoproteolytic processing of NLRP1 in the ‘function to find domain’ (FIIND) is required for inflammasome formation as it generates the FIIND UPA -CARD fragment, which can recruit ASC to initiate the inflammasome response (D’Osualdo et al, 2011; Finger et al, 2012; Gong et al, 2021; Robert Hollingsworth et al, 2021; Sandstrom et al, 2019). In steady-state, FIIND UPA -CARD remains associated with the N-terminus; FIIND UPA -CARD released from the N-terminus can also be sequestered by association with DPP9 (Hollingsworth et al, 2021; Huang et al, 2021; Okondo et al, 2018; Zhong et al, 2018).…”

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

“…The ASC PYD stem (green) with the CARD buds (orange) constitute the adaptor to Caspase-1 effector filament with the Casp-1 CARD filament (red) and the zymogen p20-p10 dimer (violet and pink) attached. The structures used to generate this model assembly were: NLRP3–NEK7 complex (6NPY(Sharif et al, 2019)), NLRP3 PYD filament (this structure), ASC PYD filament (3J63 (Lu et al, 2014)) with an overlay with the ASC full length NMR structure on the PYD subunit (2KN6 (de Alba, 2009); 20 models), ASC CARD buds (assembly of 1 or 2 ASC CARD rings on the ASC PYD stem) (6N1H (Li et al, 2018)), ASC CARD –Casp1 CARD transition (7KEU (Robert Hollingsworth et al, 2021)), Casp1 CARD filament (5FNA (Lu et al, 2016)), and Casp1-zymogen p20-p10 dimer (3E4C (Elliott et al, 2009)).…”

“…The arrangement of the subunits in these buds was modelled by using the ASC CARD filament structure (6N1H) (Li et al, 2018) as a template. A superposition of the ASC CARD to CASP1 CARD transition structure (7KEU) (Robert Hollingsworth et al, 2021) onto the buds created the transition to the CASP1 CARD filament (5FNA) (Lu et al, 2016). Finally, structural models of the CASP1-zymogen (p20-p10 dimer; 3E4C) (Elliott et al, 2009) were placed at each junction of two CASP1 CARD molecules in the filament.…”