Alejandro Rodriguez Gama1, Tejbir Kandola1, Shriram Venkatesan1, Jianzheng Wu1,2, Minling Hu1, and Randal Halfmann1,2 1Stowers Institute for Medical Research, Kansas City, MO, USA, 2Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA
Multiple signaling proteins of the innate immune system exert their cellular activities by assembling into large macromolecular complexes known as signalosomes. We previously discovered that two such proteins – the pyroptosome scaffold ASC, and the antiviral signaling protein MAVS – each assemble through self-templating polymerization that is reminiscent of infectious protein particles known as prions 1. More recently we revealed that prion-like activity broadly arises from structurally encoded kinetic barriers to nucleation – the probabilistic formation of a self-templating multimer de novo 2. For proteins like MAVS and ASC, the nucleation barrier is so high that their soluble inactive states persist despite physiological concentrations that are highly supersaturated with respect to the assembled active state. To identify other innate immune signaling proteins that may function in this manner, we used DAmFRET, a flow cytometric cell-based assay of nucleation barriers 2, to screen 138 candidate prion-like modules from 129 human proteins that function in programmed cell death and innate immune signaling. We discovered 36 of these proteins that are inherently capable of supersaturation and switch-like self-templating activation in living cells. We have further discovered a network of nucleating interactions between them, wherein polymerization of one protein nucleates the polymerization of specific additional proteins. This widespread kinetic control over cell fate indicates that cells are literally waiting to die -- pyroptosis, necroptosis, and alternative cell fates downstream of these proteins are thermodynamically favored, and therefore inevitable with time. I will discuss our investigations into the implications of this phenomenon for aging-associated inflammation and innate immune memory in human monocytes. 1. Cai, X. et al. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell 156, 1207–1222 (2014). 2. Khan, T. et al. Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior. Mol. Cell 71, 155-168.e7 (2018).