Investigating strategies to degrade the inflammasome signalling adaptor ASC

Inflammation is a vital component of host defence, but its dysregulation can lead to chronic inflammation and pathological conditions. Central to inflammatory signalling are inflammasomes, large, multiprotein cytosolic complexes that facilitate the release of pro-inflammatory cytokines IL-1β and IL-18. In many diseases, multiple inflammasomes are activated. Most of them utilise the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which serves as a crucial site of signal amplification. This makes ASC an attractive target to control multiple inflammasomes at once. Here, we explore the best strategies one could use to degrade ASC and block inflammasome signalling.To accomplish this, we engineered a functional ASC-HaloTag (ASC-HT) fusion protein, enabling selective covalent labelling and manipulation via small-molecule Halo-reactive probes. We expressed ASC-HT in ASC-deficient bone marrow-derived macrophages or human HEK293T cells and explored the best strategy to degrade ASC by leveraging HaloTag-compatible degraders. Using a proteasome-dependent degrader (Halo-PROTAC), we achieved targeted degradation of ASC-HT in both HEK293T cells and in BMDMs, which then lowered the inflammasome assembly and downstream inflammatory cytokine secretion and lytic death in BMDMs. Autophagy-dependent degrader Halo-AUTAC was not found to degrade ASC, regardless of whether ASC was expressed as a monomer or a speck-forming oligomer.Our approach provides a framework to rapidly investigate the effective degradation strategies for ASC. It also highlights ASC degradation as a promising approach for modulation of inflammasome signalling.