Inflammasomes are multiprotein complexes pivotal in the innate immune system, sensing pathogenic threats and triggering inflammatory responses. Dysregulation of inflammasomes can lead to severe autoimmune disorders. Two key DNA sensors, AIM2 and IFI16, detect pathogenic DNA through their HIN domains and mediate inflammasome assembly via their pyrin (PYD) domains, which facilitate self-association and the recruitment of the adaptor protein ASC. IFI16β, a novel human isoform of IFI16 lacking the PYD domain, has been shown in cells to bind DNA and inhibit AIM2 inflammasome formation and activity. To elucidate the inhibitory mechanism of IFI16β at the single-molecule level, we have used optical tweezers combined with confocal fluorescence microscopy to investigate AIM2’s interaction with DNA in the presence of IFI16β. Our findings reveal that IFI16β, like AIM2, binds DNA non-specifically but competes with AIM2 by significantly reducing AIM2-DNA interactions at equimolar concentration. While AIM2 binds DNA and shows little diffusion, IFI16β exhibits significant one-dimensional diffusion along DNA, suggesting that this dynamic behavior disrupts AIM2-DNA interactions. Additionally, IFI16β possesses two HIN domains connected by a long linker, potentially increasing its DNA occupancy compared to AIM2, which has only a single HIN domain. These structural and dynamic differences likely contribute to IFI16β’s inhibitory role. Our study offers new insights into the binding behavior of IFI16β, emphasizing its role in regulating AIM2 inflammasome activity through competitive DNA binding and dynamics. These findings enhance our understanding of DNA sensor regulation and offer potential implications for developing therapies targeting inflammasome-related diseases.
Bhat et al. (Sun,) studied this question.