NF-κB(核因子-κB)家族蛋白是多功能转录因子,在细胞发育中起着至关重要的调节作用。增长,凋亡,炎症,和免疫反应。然而,关于棘皮动物中这些关键基因的功能研究有限。在这项研究中,在海胆Strongylocentrotusintermedius中鉴定出NF-κB家族基因(SiRel)。该基因具有1809bp的开放阅读框长度并且编码602个氨基酸。结构域预测结果表明,SiRel蛋白的N端编码保守的Rel同源结构域(RHD),包括RHD-DNA结合域和RHD-二聚化域。多序列比较结果表明,这两个结构域的蛋白质序列是保守的。系统发育分析表明,SiRel与其他棘皮动物的P65蛋白和Rel蛋白成簇。定量实时PCR的结果表明,在所有测试的海胆组织中可检测到SiRelmRNA表达,在the中发现的最高表达水平。LPS(脂多糖)和聚(I:C)(聚肌苷酸:聚胞嘧啶)刺激后显着诱导了SiRelmRNA的表达水平。此外,SiRel卵白表达可见于HEK293T细胞的胞浆和胞核中。免疫共沉淀结果显示,SiRel能与海胆IκB(NF-κB抑制剂)蛋白相互作用。Western印迹和双荧光素酶报告基因检测结果表明,SiRel在HEK293T细胞中的过表达可以影响JNK(c-JunN末端激酶)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HEK293T细胞中的信号转导和转录激活因子3(STAT3)报告基因。总之,本研究揭示了SiRel在海胆的先天免疫应答中起着重要作用,丰富了我们对比较免疫学理论的理解。
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.