School of Agriculture, Ludong University, Yantai, 264025, China.
School of Agriculture, Ludong University, Yantai, 264025, China.
Fish Shellfish Immunol. 2024 Sep;152:109764. doi: 10.1016/j.fsi.2024.109764. Epub 2024 Jul 14.
NF-κB (Nuclear factor-kappa B) family proteins are versatile transcription factors that play crucial regulatory roles in cell development, growth, apoptosis, inflammation, and immune response. However, there is limited research on the function of these key genes in echinoderms. In this study, an NF-κB family gene (SiRel) was identified in sea urchin Strongylocentrotus intermedius. The gene has an open reading frame length of 1809 bp and encodes for 602 amino acids. Domain prediction results revealed that the N-terminal of SiRel protein encodes a conserved Rel homology domain (RHD), including the RHD-DNA binding domain and the RHD-dimerization domain. Multiple sequence comparison results showed that the protein sequences of these two domains were conserved. Phylogenetic analysis indicated that SiRel clustered with Strongylocentrotus purpuratus p65 protein and Rel protein of other echinoderms. Results from quantitative real-time PCR demonstrated detectable SiRel mRNA expression in all tested sea urchin tissues, with the highest expression level found in the gills. And SiRel mRNA expression levels were significantly induced after LPS (Lipopolysaccharide) and poly(I:C) (Polyinosinic:polycytidylic acid) stimulation. In addition, SiRel protein expression can be found in cytoplasm and nucleus of HEK293T cells. Co-immunoprecipitation results showed that SiRel could interact with sea urchin IκB (Inhibitor of NF-κB) protein. Western blotting and dual-luciferase reporter gene assay results indicated that overexpression of SiRel in HEK293T cells could impact the phosphorylation levels of JNK (c-Jun N-terminal kinase) and Erk1/2 (Extracellular signal-regulated kinases1/2) and activate interleukin-6 (IL-6), activating protein 1 (AP-1), interferon (IFN)α/β/γ, and signal transducer and activator of transcription 3 (STAT3) reporter genes in HEK293T cells. In conclusion, this study reveals that SiRel plays an important role in the innate immune response of sea urchins and enriches our understanding of comparative immunology theory.
NF-κB（核因子-κB）家族蛋白是多功能转录因子，在细胞发育、生长、凋亡、炎症和免疫反应中发挥着关键的调节作用。然而，目前关于这些关键基因在棘皮动物中的功能研究还很有限。在这项研究中，我们在海胆 Strongylocentrotus intermedius 中鉴定到了一个 NF-κB 家族基因（SiRel）。该基因的开放阅读框长度为 1809bp，编码 602 个氨基酸。结构域预测结果显示，SiRel 蛋白的 N 端编码一个保守的 Rel 同源结构域（RHD），包括 RHD-DNA 结合域和 RHD 二聚化结构域。多序列比较结果表明这两个结构域的蛋白序列具有保守性。系统进化分析表明，SiRel 与 Strongylocentrotus purpuratus p65 蛋白和其他棘皮动物的 Rel 蛋白聚类在一起。定量实时 PCR 结果表明，在所有检测的海胆组织中都可检测到 SiRel mRNA 的表达，其中在鳃中表达水平最高。LPS（脂多糖）和 poly(I:C)（聚肌胞苷酸）刺激后，SiRel mRNA 表达水平显著上调。此外，SiRel 蛋白表达可在 HEK293T 细胞的细胞质和细胞核中被发现。免疫共沉淀结果表明，SiRel 可以与海胆 IκB（NF-κB 抑制剂）蛋白相互作用。Western blotting 和双荧光素酶报告基因检测结果表明，在 HEK293T 细胞中过表达 SiRel 可影响 JNK（c-Jun N 端激酶）和 Erk1/2（细胞外信号调节激酶 1/2）的磷酸化水平，并激活白细胞介素 6（IL-6）、激活蛋白 1（AP-1）、干扰素（IFN）α/β/γ 和信号转导和转录激活因子 3（STAT3）报告基因。综上所述，本研究揭示了 SiRel 在海胆先天免疫反应中发挥着重要作用，丰富了我们对比较免疫学理论的理解。
