PT - JOURNAL ARTICLE AU - Humayun Sharif AU - L. Robert Hollingsworth AU - Andrew R. Griswold AU - Jeffrey C. Hsiao AU - Qinghui Wang AU - Daniel A. Bachovchin AU - Hao Wu TI - Structural mechanism of CARD8 regulation by DPP9 AID - 10.1101/2021.01.13.426575 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.01.13.426575 4099 - http://biorxiv.org/content/early/2021/01/14/2021.01.13.426575.short 4100 - http://biorxiv.org/content/early/2021/01/14/2021.01.13.426575.full AB - CARD8 is a germline-encoded pattern recognition receptor that detects intracellular danger signals. Like the related inflammasome sensor NLRP1, CARD8 undergoes constitutive autoprocessing within its function-to-find domain (FIIND), generating two polypeptides that stay associated and autoinhibited. Certain pathogen- and danger-associated activities, including the inhibition of the serine dipeptidases DPP8 and DPP9 (DPP8/9), induce the proteasome-mediated degradation of the N-terminal (NT) fragment, releasing the C-terminal (CT) fragment to form a caspase-1 activating inflammasome. DPP8/9 also bind directly to the CARD8 FIIND, but the role that this interaction plays in CARD8 inflammasome regulation is not yet understood. Here, we solved several cryo-EM structures of CARD8 bound to DPP9, with or without the DPP inhibitor Val-boroPro (VbP), which revealed a ternary complex composed of one DPP9, the full-length CARD8, and one CARD8-CT. Through structure-guided biochemical and cellular experiments, we demonstrated that DPP9’s structure restrains CARD8-CT after proteasomal degradation. Moreover, although DPP inhibitors do not directly displace CARD8 from DPP9 in vitro, we show that they can nevertheless destabilize this complex in cells. Overall, these results demonstrate that DPP8/9 inhibitors cause CARD8 inflammasome activation via at least two distinct mechanisms, one upstream and one downstream of the proteasome.Competing Interest StatementH.W. is a co-founder of Ventus Therapeutics. The other authors declare no competing financial interests