Functional analysis of the macromolecular inflammasome complex

The innate immune system forms the first line of defense against intruding pathogens. A core element of this response is the formation of the inflammasome, a multiprotein complex. This results in the activation of caspase-1 that leads to gasdermin-D-dependent pyroptosis, a pro-inflammatory form of cell death, and the maturation and release of the cytokines IL (interleukin)-1b and IL-18. Inflammasome formation is initiated by the activation of cytosolic pattern recognition receptors in response to various pathogenderived stimuli. Most inflammasome-forming receptors contain a PYD (Pyrin domain) through which they recruit an adaptor protein called ASC (apoptosis associated speck like protein containing a CARD). ASC consist of an N-terminal PYD and a C-terminal CARD (caspase recruitment and activation domain) through which it recruits caspase-1. A hallmark of inflammasome activation is the formation of the ASC speck, a micrometersized complex formed by ASC. It has been shown that isolated PYDs of human ASC spontaneously form a three-stranded helical filament in which the individual subunits interact with each other via six asymmetric interaction interfaces. Yet, the necessity of oligomeric ASC assemblies for its function has not been shown.
By using a combined solid-state NMR and cryo-electron microscopy approach, we show that also murine ASCPYD filaments display the same three-stranded helical arrangement as human ASCPYD filaments. This indicates that the assembly mechanism of inflammasomes is conserved amongst different species. Furthermore, we provide evidence that, within an ASC speck, filaments of ASCPYD are further condensed via ASCCARD/ASCCARD interactions. Mutations within the ASCCARD that disrupt this interaction lead to the formation of filaments instead of specks. Mutated ASCCARDs are dominant and prevent formation of dense wild type ASC specks in a dose dependent manner when expressed ectopically.
To unravel the biological role of ASC speck formation we used a structure-guided mutagenesis approach targeting all six interaction interfaces based on the murine ASCPYD filament structure. The results suggest that the ASC speck is required for efficient cytokine maturation but dispensable for pyroptosis. Specifically, the mutations E80R and Y59A in the ASCPYD either completely abrogate or reduce the speed of ASCPYD filament formation in vitro and prevent ASC speck formation in cells. These mutants still interact with PYD-containing receptors and are therefore able to support induction of pyroptosis. However, only a small amount of caspase-1 is activated which is not sufficient for efficient cytokine processing. Therefore, we conclude that the ASC speck serves as signal amplification step for inflammasome-dependent cytokine maturation.