Biochemical diversity of interferon signaling pathways in innate immunity

Abstract

Innate immune cells within mammalian organisms function as the first line of defense against infectious threats. Phagocytes, such as macrophages, respond to microbial insults by sensing pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) and activating downstream signal transduction proteins. These signaling pathways are tightly regulated to trigger various inflammatory responses, which activate the adaptive immune system and promote the resolution of infection or damage. The host interferon response represents one such system that is central to antiviral immunity. The expression of type I interferons is governed by upstream PRRs that nucleate oligomeric supramolecular organizing centers (SMOCs) via adaptor proteins including STING, TRIF, and MAVS. These adaptors utilize a conserved pLxIS motif to activate the downstream kinase TBK1 and transcription factor IRF3. Apart from the established roles of STING, TRIF, and MAVS in IRF3 activation, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Leveraging a synthetic biology-based platform to isolate and dissect pLxIS motif activities, we revealed an unexpected diversity and specificity in signaling mechanisms. This system enabled us to identify two orphan proteins that utilize pLxIS motifs, in conjunction with contiguous motifs in the same domain, to stimulate interferon responses independently of the established pathways. These two proteins, IRSp53 and GMIP, may regulate interferon signaling pathways within fibroblasts. Another interferon adaptor protein, TASL, employs an alternative mechanism whereby its pLxIS motif is physically separated from the requisite kinase activation motif downstream of MyD88. Further mechanistic analysis uncovered variable functions of each pLxIS-containing domain in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. This diversification enabled subsets of pLxIS-containing proteins to confer protection against viral infection in human cells. Overall, these collective findings establish pLxIS-containing domains as commonly used and biochemically flexible regulators of interferons and metabolism. They furthermore underscore the value of synthetic biology as a tool to discover additional regulators of innate immunity.