Multiple signaling proteins of the human innate immune system execute cell fate decisions by assembling into large macromolecular complexes, known as signalosomes, when stimulated by pathogen- or danger-associated molecules. Recent observations that some signalosome assemblies can perpetuate themselves in prion-like fashion in vivo suggest that their activation -- and the corresponding cell fate decisions -- may be thermodynamically pre-determined. In other words, signalosome assembly is inevitable, but postponed in the absence of stimulation by structurally-encoded nucleation barriers to self-assembly. We explored this hypothesis using DAmFRET, a newly developed flow cytometric assay for prion-like protein phase behavior, to identify nucleation barriers that govern self-assembly by homotypic interaction domains of the death fold, TIR, and RHIM families from over one-hundred human innate immune signaling proteins. We discovered thirty-six proteins with robust prion-like activity, including the major signalosome scaffolding proteins, or “adaptors”. We further demonstrate for a subset of the corresponding full length proteins that nucleation barriers also control the endogenous proteins’ activities in human THP-1 monocytes, and that hyper- and hypo-activating disease-causing mutations respectively decrease or increase the barriers. We provide a logic for prion-like activity in the architecture of innate immune signaling pathways, and conclude that adaptor protein supersaturation functions to store potential energy and then release it upon nucleation for rapid and effectively irreversible switch-like control over innate immunity signaling.
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