The versatile TRAF3 from sea urchin (Strongylocentrotus intermedius) interacts with MyD88 and regulates host immunity

Tumor necrosis factor receptor-associated factor 3 (TRAF3) serves as a versatile protein in innate immune signaling cascades, playing a pivotal role in modulating inflammatory responses, cellular homeostasis, and anti-infection immunity. Here, we cloned and identified a novel TRAF3 homolog (SiTRAF3) from the sea urchin Strongylocentrotus intermedius. Sequence analysis revealed that the open reading frame (ORF) of SiTRAF3 is 1914 bp in length, encoding a polypeptide of 637 amino acids. Structural prediction indicated the presence of a typical N-terminal RING finger domain, zinc finger motifs, and a highly conserved C-terminal MATH domain. Phylogenetic analysis demonstrated that SiTRAF3 clustered closely with the TRAF3-like protein from the sea urchin Strongylocentrotus purpuratus, underscoring the evolutionary conservation of TRAF3 among echinoderms. Quantitative reverse transcription PCR (qRT-PCR) assays revealed ubiquitous expression of SiTRAF3 transcripts across various tissues of S. intermedius, with the highest levels detected in the gill and coelomocytes. Following immune challenges with lipopolysaccharide (LPS), poly(I:C), and peptidoglycan, SiTRAF3 mRNA expression was significantly upregulated, suggesting an active, broad-spectrum response to diverse pathogenic stimuli. In addition, RNA interference of SiTRAF3 suppresses the expression of IL-17 family members and strongylocins following LPS stimulation. Co-immunoprecipitation (Co-IP) assays further verified a direct physical interaction between SiTRAF3 and the core adaptor protein SiMyD88. Functional analyses in HEK293T cells demonstrated that ectopic expression of SiTRAF3 notably elevated the phosphorylation levels of JNK, Erk1/2, and p38 MAPKs, and synergistically enhanced LPS-induced signaling activation. Furthermore, dual-luciferase reporter (DLR) assays revealed the broad-spectrum transcriptional activation capacity of SiTRAF3, as it significantly drove the promoter activities of nuclear factor kappa-B, activator protein-1, signal transducer and activator of transcription 3, along with multiple inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6) and interferon-related elements (interferon α/β/γ, interferon-stimulated response element). Taken together, these findings identify SiTRAF3 as a pivotal scaffold protein in the echinoid innate immune system. By assembling a signaling complex with SiMyD88, SiTRAF3 mediates the activation of MAPK cascades and multiple downstream immune transcriptional pathways. This study not only elucidates the functional significance of SiTRAF3 within the invertebrate immune regulatory network but also provides a theoretical foundation for the molecular breeding of disease-resistant sea urchin strains.