A nucleation barrier spring-loads the CBM signalosome for binary activation

Abstract

Immune cells activate in binary, switch-like fashion via large protein assemblies known as signalosomes, but the molecular mechanism of the switch is not yet understood. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, which governs NF-κB activation in both innate and adaptive immunity. We found that the switch consists of a sequence-encoded and deeply conserved nucleation barrier to ordered polymerization by the adaptor protein BCL10. The particular structure of the BCL10 polymers did not matter for activity. Using optogenetic tools and single-cell transcriptional reporters, we discovered that endogenous BCL10 is functionally supersaturated even in unstimulated human cells, and this results in a predetermined response to stimulation upon nucleation by activated CARD multimers. Our findings may inform on the progressive nature of age-associated inflammation, and suggest that signalosome structure has evolved via selection for kinetic rather than equilibrium properties of the proteins.

Protein supersaturation powers innate immune signaling

“…Indeed, soluble positive feedback mechanisms abound in biology ( 22 ), and the autocatalytic potential of pro-caspase activation in particular, would seem to obviate the prion-like mechanism for amplification. Hypothetical scaffolding functions can be largely dismissed by the fact that arbitrarily condensing or even dimerizing effectors suffices for their activation, as has been well-established for apical pro-caspases ( 23, 24 ) and effector kinases ( 25, 26 ), suggesting that the ordered structures of the polymers are materially irrelevant for activation. Indeed, even MAVS, a DFD protein with well-characterized prion-like behavior ( 13, 27 ), failed to form detectable polymers when stimulated endogenously ( 28 ).…”

“…Our recent investigation of the CBM signalosome, which regulates proinflammatory transcription factor NF-kB activation, suggests a plausible function for prion-like polymerization. We showed that the high entropic cost of the underlying transition in both density and ordering ( 5, 32, 33 ) creates a nucleation barrier large enough for the DFD adaptor protein, BCL10, to exist in a constitutively supersaturated state that stores energy for subsequent switch-like and decisive activation ( 24 ). The more phase separation is coupled to conformational ordering, the more nucleation is rate-limited by intramolecular fluctuations.…”

“…In seventeen cases the cells acquired FRET discontinuously, switching semi-stochastically to a discrete high FRET state over a range of concentrations. We previously showed that discontinuous transitions result from large sequence-encoded nucleation barriers that are characteristic for prion-like self-assembly, whereas continuous transitions instead reflect phase separation through dynamic low-affinity interactions ( 24, 32, 41, 42 ).…”

“…Ordered self-assemblies tend to deviate from sphericity, and some DFDs have been found to polymerize into long filaments in the cytoplasm ( 14, 24, 43 ). We therefore assessed the subcellular distribution of each protein using high-throughput confocal microscopy.…”

Chimeric and mutant CARD9 constructs enable analyses of conserved and diverged autoinhibition mechanisms in the CARD-CC protein family

“…1a) [2], [10], [11], forming the CARMA subfamily. A common theme between the four jawed vertebrate CARD-CC proteins is that they all get activated by protein kinase C (PKC) [11], and will subsequently form filamentous polymeric filaments by aggregation via their CC domain [4], [12]- [15]. These CARD-CC filaments will bind the signaling proteins BCL10 and MALT1 via the CARD domain, which subsequently will signal towards NF-κB activation and activation of the paracaspase MALT1 protease activity [16]- [21].…”