Distinct interaction modes orchestrate the polymerization of ASC PYD and CARD domains into filamentous structures.

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC plays a key role in the inflammasome complex by forming a supramolecular structure called the ASC speck, which promotes inflammation and pyroptosis. The assembly of ASC-dependent inflammasomes is mediated by homotypic interactions between receptor, adaptor, and effector proteins, with PYD-PYD and CARD-CARD interactions categorized into three major types (type I, II, and III). These interactions orchestrate the homo-oligomerization of inflammasome components, serving as a platform for caspase-1 activation. Through maintaining interaction homeostasis, ASC regulates innate immune responses and functions as a tumor suppressor. Dysregulation of ASC due to genetic mutations is implicated in various cancers and autoimmune diseases. However, the mechanisms driving ASC speck formation remain unclear, leaving questions on its domain-specific interactions. To address this, we used a cell line model to investigate the roles of single and double mutations within the PYD and CARD domains of ASC. We separately fused wild-type (wt)PYD and wtCARD domains to GFP and mCherry to assess the effects of these mutations on interaction dynamics using fluorescence microscopy and Förster resonance energy transfer (FRET) systems. Our results reveal previously unknown cooperative mechanisms in which specific PYD and CARD residues function as enhancers or disruptors of homo-oligomerization, highlighting the importance of cumulative interaction effects. Our study provides new insights into the molecular basis of ASC domain polymerizations.