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TNFR-associated factors (TRAFs) and receptor-interacting protein kinases (RIPKs) are important immunological linker molecules in mammals and play important roles in the TNFα, TLR and IFN signaling pathways. However, the evolutionary origins of these genes in vertebrates have not previously been described in lampreys. In this study, we searched the genomes of  Lampetra japonicum, Lethenteron reissneri , and  Petromyzon marinus  for genes encoding  trafs  and  ripks  and performed homologous sequence alignment, phylogenetic tree, functional domain, conserved motif, gene structure, and synteny analyses to determine their evolutionary relationships. The distribution of the lamprey  traf  and  ripk  families and the immune response of the gene families in lampreys stimulated by different pathogens were also demonstrated, suggesting a role of structural changes in expression and functional diversification. Additionally, the dual luciferase reporter gene assay showed that the addition of exogenous immunomodulator (TNFα or IFN) to the overexpression of LjLRIPK1a or LjTRAF3/6 significantly downregulated NF-κB or ISRE activation. LjRIPK1a can significantly enhance caspase-8 activity, and overexpression of LjRIPK1a or LjTRAF3a/6 in HEK293T cells results in cell apoptosis. In summary, this study makes an important contribution to the understanding of the  traf  and  ripk  gene families in different vertebrates. Our results also provide new evidence for the evolution of vertebrate TRAFs and RIPKs and their impacts on immune regulation.

frontiers in immunology

Comprehensive Evolutionary Analysis of Lamprey TNFR-Associated Factors (TRAFs) and Receptor-Interacting Protein Kinase (RIPKs) and Insights Into the Functional Characterization of TRAF3/6 and RIPK1