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.
Xiaolong Chu, Fengchen Liu, Yingying Liu et al.· Fish and Shellfish Immunolog...· 0 citations
Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an atypical member of the TRAF family, with well-established roles in modulating vertebrate innate immunity, apoptosis, and intracellular signal transduction. In contrast, its functional repertoire in invertebrates, particularly among marine mollusks, remains poorly characterized. In this study, a TRAF7 ortholog from the Zhikong scallop (Chlamys farreri) was identified and designated as CfTRAF7. The cloned CfTRAF7 cDNA comprises a 1953-bp open reading frame encoding a 650-amino-acid polypeptide. Function domain prediction confirmed that the N-terminal RING finger domain and the C-terminal WD40 repeat region exhibit significant sequence homology with TRAF7 orthologs across metazoans. Phylogenetic analysis indicated that CfTRAF7 clusters with TRAF7 homologs from other mollusks. Quantitative real-time PCR assays demonstrated that CfTRAF7 mRNA was widely expressed in all tested scallop tissues, with the highest expression in hemocytes. After stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid, and peptidoglycan, the mRNA expression of CfTRAF7 was significantly upregulated, with temporal differences in response to various pathogen-associated molecular patterns. Co-immunoprecipitation confirmed that CfTRAF7 interacted with C. farreri transforming growth factor beta-activated kinase 1 (CfTAK1). Western blot analysis revealed that overexpression of CfTRAF7 in HEK293T cells significantly enhanced the phosphorylation of mitogen-activated protein kinase (MAPK) proteins (e.g. JNK and Erk1/2), whereas RNA interference (RNAi) of CfTRAF7 suppressed MAPK phosphorylation levels in vivo following LPS challenge, but had no obvious effect on p38 phosphorylation. RNAi assays demonstrated that silencing of CfTRAF7 significantly altered the expression of several immune-related genes, including CfTAK1, CfRel, CfIL17A, CfIL17D, CfTNF-like, and CfSOD, following LPS challenge. Dual-luciferase reporter assay showed that overexpression of CfTRAF7 could activate immune-related reporter genes such as interleukin-6 (IL-6), interferon α/γ (IFNα/γ), activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), and tumor necrosis factor α (TNFα), in a dose-dependent manner. This study not only enriches the functional research on TRAF7 in invertebrates but also provides a new perspective for understanding the evolutionary conservation and species specificity of molluscan innate immune signaling pathways. Additionally, it also offers important theoretical support for further comprehension of the molecular mechanism of scallop immune defense and promoting the green control of aquatic diseases in marine farming.
Xiaoxue Lin, Zihao Zhang, Xiaolong Chu et al.· Fish and Shellfish Immunolog...· 0 citations