Novel Findings in Teleost TRAF4, a Protein Acts as an Enhancer in TRIF and TRAF6 Mediated Antiviral and Inflammatory Signaling

Abstract

Tumor necrosis factor receptor-associated factors (TRAFs) are important adaptor molecules that play important roles in host immune regulation and inflammatory responses. Compared to other members of TRAFs, the function of TRAF4 in vertebrate immunity remains unclear, especially in teleosts. In the present study, TRAF4 ortholog was cloned and identified in large yellow croaker (Larimichthys crocea), named as Lc-TRAF4. The open reading frame (ORF) of Lc-TRAF4 is 1,413 bp and encodes a protein of 470 amino acids (aa), which is consisted of a RING finger domain, two zinc finger domains, and a MATH domain. The genome organization of Lc-TRAF4 is conserved in teleosts, amphibians, birds, and mammals, with 7 exons and 6 introns. Quantitative real-time PCR analysis revealed that Lc-TRAF4 was broadly distributed in various organs/tissues of healthy large yellow croakers and could be significantly up-regulated in the gill, intestine, spleen, head kidney, and blood under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations. Notably, luciferase assays showed that overexpression of Lc-TRAF4 could significantly induce the activation of IRF3, IRF7, and type I IFN promoters, with the RING finger and zinc finger domains function importantly in such promoter activation. Confocal microscopy revealed that Lc-TRAF4 is located in the cytoplasm, whereas the deletion of the RING finger, zinc finger or MATH domain showed little effect on the subcellular localization of Lc-TRAF4. Interestingly, Lc-TRAF4 overexpression could significantly enhance Lc-TRIF and Lc-TRAF6 medicated IRF3 and IRF7 promoter activation. In addition, co-expression of Lc-TRAF4 with Lc-TRIF or Lc-TRAF6 could significantly induce the expression of antiviral and inflammation-related genes, including IRF3, IRF7, ISG15, ISG56, Mx, RSAD2, TNF-α, and IL-1β compared to the only overexpression of Lc-TRAF4, Lc-TRIF or Lc-TRAF6. These results collectively imply that Lc-TRAF4 functions as an enhancer in Lc-TRIF and Lc-TRAF6 mediated antiviral and inflammatory signaling.

Teleost TRAF7, a protein functions in the host antiviral responses via NF-κB and IRF3/7 mediated signaling

“…The cells were then kept in an incubator at 37°C and 5% CO 2 as our previous reports (Zou et al, 2019;Zou et al, 2020). Large yellow croaker muscle (LYCMS) cells were maintained in L15 Medium (Boster, USA) containing 10% FBS and 100 U/mL PS at 27°C as our previous report (Chen et al, 2022). Epithelioma papulosum cyprinid (EPC) cells were cultured at 25°C in M199 medium (Procell, Wuhan, China) containing 10% FBS and 100 U/mL PS.…”

“…After that, the cells were collected at 48 h post-transfection (hpt), with the expression patterns of TRAF7 and the antiviral and inflammatory-related genes mentioned above detected by qRT-PCR analysis. All primers used are listed in Table 1 and could also refer to our previous reports (Chen et al, 2022;Luo et al, 2022).…”

Structural Characterization of TRAF6 N-Terminal for Therapeutic Uses and Computational Studies on New Derivatives

“…They are the main signal transducers for the TNF receptor, the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR), and NOD-like receptor (NLR) superfamilies. Until recently, TRAFs were classified as classical members (TRAF1-6) and a single nonclassical member (TRAF7) [5][6][7]. In general, TRAFs (TRAF1-7) adopt a common structure including the Really Interesting New Gene (RING) domain (except for TRAF1), zinc finger motifs, a coiled-coil domain, and a highly conserved C-terminal β-sandwich domain (TRAF-C or MATH domain).…”