1.
Protein supersaturation powers innate immune signaling.
Abstract:
Innate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturated in vivo. Our findings reveal that the soluble “inactive” states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.
2.
A nucleation barrier spring-loads the CBM signalosome for binary activation.
Abstract:
Immune cells activate in a binary, switch-like fashion that involves proteins polymerizing into large complexes known as signalosomes. The switch-like nature of signalosome formation has been proposed to result from large energy barriers to polymer nucleation. Whether such nucleation barriers indeed drive binary immune responses has not yet been shown. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, a key determinant of transcription factor NF-κB activation in both innate and adaptive immunity. We found that the adaptor protein BCL10 encodes an intrinsic nucleation barrier, and that this barrier has been conserved from cnidaria to humans. Using optogenetic tools and a single-cell transcriptional reporter of NF-κB activity, we further revealed that endogenous human BCL10 is supersaturated even in unstimulated cells, indicating that the nucleation barrier operationally stores energy for subsequent activation. We found that upon stimulation, BCL10 nucleation by CARD9 multimers triggers self-templated polymerization that saturates NF-κB activation to produce a binary response. Pathogenic mutants of CARD9 that cause human immunodeficiencies eliminated nucleating activity. Conversely, a hyperactive cancer-causing mutation in BCL10 increased its spontaneous nucleation. Our results indicate that unassembled CBM signalosome components function analogously to a spring-loaded mousetrap, constitutively poised to activate NF-κB through irrevocable polymerization. This finding may inform our understanding of the root causes and progressive nature of pathogenic and age-associated inflammation.
