Abstract
Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.
Highlights
Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex
We investigate the structural basis of how the auto-proteolysis-dependent, activating domains of human NLRP1 and CARD8 (FIINDUPA-caspase activation and recruitment domain (CARD)) trigger inflammasome assembly and activation
We and others have demonstrated that Cterminal auto-cleavage fragments of NLRP1 and CARD8 (NLRP1-FIINDUPA-CARD and CARD8-FIINDUPA-CARD) are sufficient to drive pyroptosis in cultured human cells[9,11,22] (Fig. 1a)
Summary
Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. We report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. We demonstrate that FIINDUPA and CARD domains play non-redundant roles in NLRP1 activation: NLRP1-CARD is sufficient to form filaments in vitro, trigger ASC speck formation and activate pyroptosis in human cells, albeit with limited efficiency. By solving the cryo-EM structures NLRP1- and CARD8-CARD core filaments at 3.7 Å, we map the precise structural motifs that mediate heterotypic interactions between the CARD domains of NLRP1, CARD8, ASC, and CASP1 This analysis provides a structural explanation as to why activated NLRP1 avidly triggers ASC speck formation, whereas CARD8 directly activates pro-caspase-1. In conjunction with three complementary[19,20,21] preprint manuscripts published while this manuscript was in revision, our findings unveil the structural basis for NLRP1 and CARD8 inflammasome assembly and suggest that intrinsic structural properties of the CARD domain can determine the specificity of inflammasome signaling
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