State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, No 7 Donghu South Road, Wuhan 430072, China.
Fish Shellfish Immunol. 2012 Sep;33(3):459-67. doi: 10.1016/j.fsi.2012.05.025. Epub 2012 May 31.
Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein-protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study, Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway.
Tollip 和 IRAK-1 是哺乳动物 TLR/IL-1R 信号通路的关键组成部分，作为 TLR/IL-1R 信号转导通路的介质发挥着至关重要的作用。虽然在鱼类中已经发现了几种 TLR，但 TLR 信号通路在鱼类感染诱导免疫中的分子关联、蛋白-蛋白相互作用或作用还很少受到关注。在本研究中，从草鱼头肾 cDNA 文库中分离出 Tollip 和 IRAK-1 序列。通过 3'和 5'RACE 分别获得全长转录本和启动子区域序列，通过报告基因-启动子构建体和实时 RT-PCR 分析确定草鱼 Tollip 和 IRAK-1 在组织中的转录模式。使用重组蛋白进行抗体生产。系统发育分析表明，草鱼的这些基因座分别与先前报道的 Tollip 和 IRAK-1 基因聚类，其序列与斑马鱼（Danio rerio）的基因具有最高的同一性。草鱼 Tollip 和 IRAK-1 的启动子区域被证明是活跃的。在病毒感染后，这两个基因座的转录水平在大多数免疫相关组织中均呈时间依赖性上调。使用本研究中产生的抗体进行免疫荧光分析表明，Tollip 和 IRAK-1 在 CIK 细胞的细胞质中均匀分布并共定位。然而，在病毒感染后，Tollip 和 IRAK-1 都有向细胞膜移动的趋势。我们的研究结果表明 Tollip 和 IRAK-1 蛋白存在于硬骨鱼类中，并表明 Tollip-IRAK-1 复合物在受到病毒刺激后被募集到受体复合物中。这些结果为 TLR 信号通路在硬骨鱼类中的作用提供了新的见解，特别是硬骨鱼类 Tollip 和 IRAK-1 的作用以及这些分子作为该通路一部分的相互作用。
