The CARD8 T60 variant associates with NLRP1 and negatively regulates its activation

The NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates the NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that different ASC preference in three types of inflammasomes, namely the ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that signal transduction of the NLRP1 inflammasome relies on multiple interaction surfaces at its CARD domain. Finally, our results partly explain how mutations in NLRP1 lead to its constitutive activation in auto-inflammatory diseases. In conclusion, our study not only reveals how CARD8 downregulates the NLRP1 inflammasome activation, but also provides insights into the assembly mechanisms of CARD-containing inflammasomes.