Newly identified TRAF7 interacts with TAK1 and regulates immune response in Chlamys farreri.

Abstract

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.