肿瘤坏死因子受体相关因子(TRAFs)是重要的衔接分子,在宿主免疫调节和炎症反应中起重要作用。与TRAF的其他成员相比,TRAF4在脊椎动物免疫中的功能尚不清楚,尤其是在硬骨鱼中。在本研究中,在大黄鱼(Larimichthyscrocea)中克隆并鉴定了TRAF4直系同源物,命名为Lc-TRAF4。Lc-TRAF4的开放阅读框(ORF)为1,413bp,编码470个氨基酸(aa)的蛋白质,它由一个RING手指域组成,两个锌指结构域,和数学域。Lc-TRAF4的基因组组织在硬骨鱼中是保守的,两栖动物,鸟,哺乳动物,有7个外显子和6个内含子。实时定量PCR分析显示,Lc-TRAF4广泛分布于健康大黄鱼的各种器官/组织中,并可在g中显著上调,肠,脾,脾头肾,和聚I:C下的血液,LPS,PGN,和斑纹假单胞菌的刺激。值得注意的是,荧光素酶检测表明,Lc-TRAF4的过表达能显著诱导IRF3、IRF7和I型IFN启动子的激活,环指和锌指结构域在这种启动子激活中起重要作用。共聚焦显微镜显示Lc-TRAF4位于细胞质中,而戒指手指的删除,锌指或MATH结构域对Lc-TRAF4的亚细胞定位影响不大。有趣的是,Lc-TRAF4过表达可显著增进Lc-TRIF和Lc-TRAF6含药IRF3和IRF7启动子的激活。此外,Lc-TRAF4与Lc-TRIF或Lc-TRAF6共表达可显著诱导抗病毒和炎症相关基因的表达,包括IRF3,IRF7,ISG15,ISG56,Mx,RSAD2,TNF-α,与仅Lc-TRAF4、Lc-TRIF或Lc-TRAF6的过表达相比。这些结果共同暗示Lc-TRAF4在Lc-TRIF和Lc-TRAF6介导的抗病毒和炎症信号传导中起增强剂的作用。
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.