Teleost-specific TLR25/MyD88 signaling promotes K63-linked ubiquitination of IRF1 to induce IFNa gene expression

Interferon regulatory factor 1 (IRF1) serves as the primary transcription factor responsible for inducing interferon a (IFNa) gene expression in teleost fish. While mammalian IRF1 is known to be unstable due to ubiquitin-mediated degradation, its function critically dependents on stability regulation by upstream signals. However, the mechanisms governing the protein stability of IRF1 remain unexplored in teleost fish. In this study, we identified IRF1 from ya-fish (Schizothorax prenanti) and demonstrated its specific role in activating IFNa transcription. Notably, teleost-specific Toll-like receptor 25 (TLR25) enhances IRF1 protein stability through the direct recruitment of the adaptor MyD88, thereby facilitating IFNa gene expression. The death domain (DD) and intermediate domain (IM) of MyD88 are essential for this stabilization. IRF1 undergoes both K48- and K63-linked ubiquitination modifications, which exhibit competitive antagonism. The TLR25/MyD88 signaling promotes both types of ubiquitination but preferentially amplifies K63-linked ubiquitination over K48-linked ubiquitination. Furthermore, this enhancement relies on TLR25/MyD88-driven activation of NF-κB inflammatory pathway. Our findings contribute to the understanding of how fish regulate IRF1 protein stability to increase IFNa gene expression during pathogen invasions.