Ring Finger protein 34 negatively regulates MyD88-mediated NF-κB signaling via the ubiquitin-proteasome pathway in miiuy croaker (Miichthys miiuy)

Innate immunity constitutes a fundamental defense mechanism in host immunity, wherein myeloid differentiation factor 88 (MyD88) functions as the central adaptor protein in Toll-like receptor (TLR) signaling pathways, orchestrating teleost innate immune responses the nuclear factor-kappa B (NF-κB) pathway. To elucidate the regulatory mechanism of E3 ligase RNF34 (Ring Finger Protein 34) in this signaling cascade, we employed miiuy croaker (Miichthys miiuy) as a model organism and conducted a series of experiments. Luciferase reporter assays demonstrated that RNF34 exerted dose- and time-dependent inhibition on the MyD88-mediated NF-κB signaling pathway. This inhibitory effect persisted under LPS stimulation, confirming RNF34's stable regulatory function. Western blot analysis further revealed that RNF34 negatively regulated MyD88 protein expression, and this regulatory effect was significantly enhanced under LPS stimulation. Mechanistic investigations showed that cycloheximide (CHX) chase assays indicated RNF34 significantly shortened MyD88 protein half-life; treatment with the proteasome inhibitor MG132 completely reversed RNF34-mediated MyD88 degradation; and ubiquitination assays demonstrated that RNF34 substantially enhanced MyD88 ubiquitination levels. These findings collectively indicate that RNF34 promotes MyD88 ubiquitination, leading to its proteasomal degradation and inhibition of NF-κB signaling pathway activation. This study enriches the understanding of RNF34 as a negative immune regulator in miiuy croaker, providing evidence for the regulatory mechanism of the NF-κB signaling pathway in teleosts and offering insights into the precise maintenance of immune homeostasis in teleost fishes.