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

Abstract

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.

CARD8: A Novel Inflammasome Sensor with Well-Known Anti-Inflammatory and Anti-Apoptotic Activity

“…More experiments are required to elucidate the molecular mechanisms underlying the regulation of caspase-1 activation induced by CARD8, as well as the factors influencing the apparent substrate specificity of the protease, which seems to depend on the recruiting inflammasome sensor. The FIIND of CARD8 acts not only as a proteolytic self-activation domain, but can also bind to, and inhibit, the activation of the inflammasome sensors NLRP3, NLRP1, and, possibly, AIM2 [ 89 , 90 ]. Therefore, a crosstalk, mostly mediated through direct interactions, seems to exist between CARD8 and the mentioned inflammasome sensors.…”

“…Overexpression experiments in HEK293 cells, reconstituted with NLRP1 inflammasome components, demonstrated that CARD8 T60, but not CARD8 T48, binds and inhibits NLRP1 activation [ 90 ]. Expression of T60 in human skin was confirmed at the transcriptional level.…”

“…Expression of T60 in human skin was confirmed at the transcriptional level. Co-immunoprecipitation experiments revealed that the ZU5 part of the FIIND of CARD8 can interact with the NACHT, LRRs, and FIIND of NLRP1 [ 90 ]. Interestingly, CARD8 T60 was found to inhibit wild-type NLRP1, but not two gain-of-function mutants of NLRP1, suggesting that these mutants may support NLRP1 activation, as their interaction with CARD8 is negatively affected [ 90 ].…”

“…Co-immunoprecipitation experiments revealed that the ZU5 part of the FIIND of CARD8 can interact with the NACHT, LRRs, and FIIND of NLRP1 [ 90 ]. Interestingly, CARD8 T60 was found to inhibit wild-type NLRP1, but not two gain-of-function mutants of NLRP1, suggesting that these mutants may support NLRP1 activation, as their interaction with CARD8 is negatively affected [ 90 ]. However, it should be considered that this interpretation is based on less physiological overexpression experiments, where the autocatalytic self-processing of CARD8 did not occur [ 89 , 90 ].…”

“…Interestingly, CARD8 T60 was found to inhibit wild-type NLRP1, but not two gain-of-function mutants of NLRP1, suggesting that these mutants may support NLRP1 activation, as their interaction with CARD8 is negatively affected [ 90 ]. However, it should be considered that this interpretation is based on less physiological overexpression experiments, where the autocatalytic self-processing of CARD8 did not occur [ 89 , 90 ]. Nevertheless, experiments in endothelial cells and keratinocytes, which express both NLRP1 and CARD8 endogenously, also suggest a crosstalk of NLRP1 and CARD8 [ 43 , 79 , 80 ].…”