Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adapter protein in the toll-like receptor signaling pathway that triggers downstream molecules involved in innate immunity. Although TRAF6 has been well studied in mammals, the molecular information and function of TRAF6 in fish is still limited. Here, we identified and analyzed a TRAF6 homolog (LmTRAF6) from the spotted sea bass (Lateolabrax maculatus). Similar to its counterparts in mammals and other fish species, LmTRAF6 shares the domain topology containing one N-terminal RING, two TRAF-type zinc fingers, a coiled-coil region and a C-terminal MATH domain. Despite a sequence similarity of 60% with mammalian TRAF6s, LmTRAF6 shares higher similarities with teleost homologs (~68%–93%). The coding region of LmTRAF6 gene contains seven exons and six introns, which is consistent to the genetic organization in grouper and rock bream, but not in zebrafish, common carp and tetrapods (the sixth intron was lost resulting in a combined exon). Quantitative real-time polymerase chain reaction analysis revealed that LmTRAF6 transcripts were ubiquitously expressed in all tested tissues and upregulated after Vibrio. harveyi and S. agalactiae infection. LmTRAF6 could assist HEK293T cells to survive by inhibiting apoptosis under both V. harveyi and S. agalactiae stimulation. Intracellular localization showed that LmTRAF6 was localized mainly in the cytoplasm. Overexpression of wild-type (WT) LmTRAF6 and the truncated form of △MATH increased the ability of NF-κB in HEK293T cells, whereas truncations, including the △RING and △coiled-coil domain, did not significantly activate NF-κB, indicating that the RING finger and coiled-coil domain play crucial roles in downstream signal transduction. In addition, overexpression of LmTRAF6-WT significantly increased the activation of NF-κB in HEK293T cells under V. harveyi and S. agalactiae stimulation. These results suggest that LmTRAF6 activates NF-κB and plays a potential role in the immune defense system against bacterial infection.

肿瘤坏死因子受体相关因子6（TRAF6）是toll样受体信号通路中的关键衔接子蛋白，可触发参与先天免疫的下游分子。尽管已经在哺乳动物中对TRAF6进行了充分的研究，但是TRAF6在鱼类中的分子信息和功能仍然有限。在这里，我们从斑点鲈鱼（Lateolabrax maculatus）中鉴定并分析了TRAF6同源物（LmTRAF6）。）。与哺乳动物和其他鱼类中的类似物一样，LmTRAF6共有一个域拓扑结构，其中包含一个N端RING，两个TRAF型锌指，一个卷曲螺旋区和一个C端MATH域。尽管与哺乳动物TRAF6s有60％的序列相似性，但LmTRAF6与硬骨鱼同源物具有更高的相似性（〜68％–93％）。LmTRAF6基因的编码区包含七个外显子和六个内含子，这与石斑鱼和鲷鱼的遗传组织一致，但与斑马鱼，鲤鱼和四足动物的遗传组织一致（第六个内含子丢失，导致一个外显子结合在一起）。实时定量聚合酶链反应分析表明，LmTRAF6转录本在所有测试组织中普遍表达，在弧菌感染后上调。哈维和无乳链球菌感染。LmTRAF6可以通过抑制哈维氏弧菌和无乳链球菌刺激下的细胞凋亡来帮助HEK293T细胞存活。细胞内定位表明LmTRAF6主要定位在细胞质中。野生型（WT）LmTRAF6的过表达和△MATH的截短形式增加了HEK293T细胞中NF-κB的能力，而截短，包括△RING和△卷曲螺旋域，并未显着激活NF-κB，表明RING指和螺旋线圈​​域在下游信号转导中起关键作用。此外，过表达LmTRAF6-WT显着增加了哈维氏弧菌和无乳链球菌对HEK293T细胞中NF-κB的活化作用。刺激。这些结果表明，LmTRAF6激活NF-κB并在抵抗细菌感染的免疫防御系统中发挥潜在作用。