Signalosome nucleation enables digital innate immune proinflammatory responses

Abstract

Pathogen sensing in innate immune cells begins with the activation of Pathogen Recognition Receptors (PRR) that result in the formation of polymeric protein self‐assemblies known as signalosomes. The proinflammatory response resulting from signalosome formation is thought to produce a binary all‐or‐none response. How this is controlled remains unclear. Because the activation of ordered self‐assemblies like signalosomes implies a rate‐limiting nucleation step, we hypothesized that a nucleation barrier imposed by the signalosome assembly enables the digital all‐or‐none response. Here we focused on the signalosome CARD9‐Bcl10‐MALT1 (CBM), which functions downstream of fungal PRR, Dectin‐1, which activates NF‐κB. We employed DAmFRET, a recently developed method that allows to determine the frequency of protein nucleation in cells using flow cytometry. We determined that CARD9 and Bcl10 form nucleation‐limited polymers in cells with critical concentrations similar to those from in vitro studies. We then dissected the nucleation steps required for signalosome formation. Co‐expression of artificial CARD9 polymer seeds with Bcl10 resulted in the complete nucleation of Bcl10 monomeric protein. Remarkably, CARD9 seeds containing mutations that impair antifungal signaling in humans were unable to nucleate Bcl10, suggesting that a direct seeding effect of CARD9 over Bcl10 is critical for its function. We next asked whether the formation of the signalosome drives the digital activation of NF‐κB. To do so, we coupled a transcriptional fluorescent NF‐κB reporter to DAmFRET. We determined that cells containing Bcl10 assemblies strongly induced NF‐κB activation, while cells expressing a similar protein concentration in the monomeric state did not. We asked next whether mutations that alter the nucleation barriers likewise modulate NF‐κB activation. Disruption of polymer formation with mutation E53R as well as increasing the critical concentration of polymerization with mutant R36E, both impaired NF‐κB activation. In contrast, mutation R58G decreased the nucleation barrier while also exacerbating the activation of NF‐κB. Our results indicate a critical role of nucleation barriers in governing the functional outcome of signalosomes. We anticipate the impact of our work could uncover the molecular mechanism of innate immune digital proinflammatory activation, which may serve as a basis to understand the cause of autoinflammatory diseases.Support or Funding InformationStowers Institute for Medical Research