School of Agriculture, Ludong University, Yantai, China.
Ocean School, Yantai University, Yantai, China.
Front Immunol. 2023 Jan 12;13:1091419. doi: 10.3389/fimmu.2022.1091419. eCollection 2022.
Inhibitor of κB kinase (IKK) family proteins are key signaling molecules in the animal innate immune system and are considered master regulators of inflammation and innate immunity that act by controlling the activation of transcription factors such as NF-κB. However, few functional studies on IKK in invertebrates have been conducted, especially in marine mollusks. In this study, we cloned the IKK gene in the Zhikong scallop and named it . encodes a 773-amino acid-long protein, and phylogenetic analysis showed that CfIKK3 belongs to the invertebrate TBK1/IKKϵ protein family. Quantitative real-time PCR analysis showed that mRNA is ubiquitously expressed in all tested scallop tissues. The expression of transcripts was significantly induced after challenge with lipopolysaccharide, peptidoglycan, or poly(I:C). Co-immunoprecipitation (co-IP) assays confirmed the direct interaction of CfIKK3 with MyD88 (the key adaptor in the TLR pathway) and MAVS (the key adaptor in the RLR pathway), suggesting that this IKK protein plays a crucial role in scallop innate immune signal transduction. In addition, the CfIKK3 protein formed homodimers and bound to CfIKK2, which may be a key step in the activation of its own and downstream transcription factors. Finally, in HEK293T cells, dual-luciferase reporter gene experiments showed that overexpression of CfIKK3 protein activated the NF-κB reporter gene in a dose-dependent manner. In conclusion, our experimental results confirmed that CfIKK3 could respond to PAMPs challenge and participate in scallop TLR and RLR pathway signaling, ultimately activating NF-κB. Therefore, as a key signaling molecule and modulator of immune activity, CfIKK3 plays an important role in the innate immune system of scallops.
IKK 家族蛋白是动物先天免疫系统中的关键信号分子，被认为是炎症和先天免疫的主要调节剂，通过控制转录因子如 NF-κB 的激活来发挥作用。然而，在无脊椎动物中，对 IKK 的功能研究很少，特别是在海洋软体动物中。在本研究中，我们克隆了中国蛤蜊中的 IKK 基因，并将其命名为 。 编码一个 773 个氨基酸长的蛋白质，系统发育分析表明，CfIKK3 属于无脊椎动物 TBK1/IKKϵ 蛋白家族。定量实时 PCR 分析表明， 在所有检测的中国蛤蜊组织中均广泛表达。 在受到脂多糖、肽聚糖或聚（I:C）的刺激后， 的转录物表达显著诱导。 免疫共沉淀（co-IP）实验证实 CfIKK3 与 MyD88（TLR 途径中的关键衔接子）和 MAVS（RLR 途径中的关键衔接子）直接相互作用，表明该 IKK 蛋白在贝类先天免疫信号转导中发挥关键作用。 此外，CfIKK3 蛋白形成同源二聚体并与 CfIKK2 结合，这可能是其自身和下游转录因子激活的关键步骤。 最后，在 HEK293T 细胞中，双荧光素酶报告基因实验表明 CfIKK3 蛋白的过表达以剂量依赖的方式激活 NF-κB 报告基因。 综上所述，我们的实验结果证实 CfIKK3 可以对 PAMPs 刺激做出反应，并参与贝类 TLR 和 RLR 途径信号转导，最终激活 NF-κB。 因此，作为关键的信号分子和免疫活性调节剂，CfIKK3 在贝类先天免疫系统中发挥重要作用。
