College of Animal Science, Guizhou University, Guiyang 550025, China.
College of Animal Science, Guizhou University, Guiyang 550025, China; Key Laboratory of Animal Genetics, Breeding and Reproduction in The Plateau Mountainous Region, Ministry of Education, Guizhou University, Guiyang 550025, China.
Poult Sci. 2024 Mar;103(3):103461. doi: 10.1016/j.psj.2024.103461. Epub 2024 Jan 12.
The speckle-type POZ protein (SPOP) is demonstrated to be a specific adaptor of the cullin-RING-based E3 ubiquitin ligase complex that participates in multiple cellular processes. Up to now, SPOP involved in inflammatory response has attracted more attention, but the association of SPOP with animal virus infection is scarcely reported. In this study, chicken MyD88 (chMyD88), an innate immunity-associated protein, was screened to be an interacting partner of chSPOP using co-immunoprecipitation (Co-IP) combined with liquid chromatography-tandem mass spectrometry methods. This interaction was further confirmed by fluorescence co-localization, Co-IP, and pull-down assays. It was interesting that exogenous recombinant protein HA-chSPOP or endogenous chSPOP alone was mainly located in the nucleus but was translocated to the cytoplasm upon co-expression with chMyD88 or lipopolysaccharide stimulation. In addition, chSPOP reduced chMyD88 expression by ubiquitination in a dose-dependent manner, and the regulation of NF-κB activity by chSPOP was dependent solely on chMyD88. Importantly, chSPOP played a negative regulatory role in the MyD88/NF-κB signaling pathway and the production of proinflammatory cytokines. Moreover, we found that velogenic Newcastle disease virus (NDV) infection changed the subcellular localization of chSPOP and the expression patterns of chSPOP and chMyD88, and overexpression of chSPOP decreased the production of proinflammatory cytokines to enhance velogenic and lentogenic NDV replication, while siRNA-mediated chSPOP knockdown obtained the opposite results, thereby indicating that chSPOP negatively regulated MyD88/NF-κB signaling pathway mediated proinflammatory cytokine production to promote NDV replication. These findings highlight the important role of the SPOP/MyD88/NF-κB signaling pathway in NDV replication and may provide insightful information about NDV pathogenesis.
斑点型 POZ 蛋白（SPOP）被证明是一种特异性衔接物，可参与基于 Cullin-RING 的 E3 泛素连接酶复合物，从而参与多种细胞过程。迄今为止，SPOP 参与炎症反应引起了更多关注，但 SPOP 与动物病毒感染的关联鲜有报道。在本研究中，通过免疫共沉淀（Co-IP）结合液相色谱-串联质谱法筛选到鸡 MyD88（chMyD88），一种先天免疫相关蛋白，是 chSPOP 的相互作用伙伴。通过荧光共定位、Co-IP 和下拉实验进一步证实了这种相互作用。有趣的是，外源性重组蛋白 HA-chSPOP 或内源性 chSPOP 单独主要位于细胞核内，但在与 chMyD88 共表达或脂多糖刺激时，会转移到细胞质中。此外，chSPOP 以剂量依赖的方式通过泛素化降低 chMyD88 的表达，并且 chSPOP 对 NF-κB 活性的调节仅依赖于 chMyD88。重要的是，chSPOP 在 MyD88/NF-κB 信号通路和促炎细胞因子的产生中发挥负调节作用。此外，我们发现，禽传染性支气管炎病毒（NDV）感染改变了 chSPOP 的亚细胞定位以及 chSPOP 和 chMyD88 的表达模式，并且过表达 chSPOP 会降低促炎细胞因子的产生，从而增强了强毒和弱毒 NDV 的复制，而 siRNA 介导的 chSPOP 敲低则获得了相反的结果，这表明 chSPOP 负调控 MyD88/NF-κB 信号通路介导的促炎细胞因子产生，从而促进 NDV 复制。这些发现强调了 SPOP/MyD88/NF-κB 信号通路在 NDV 复制中的重要作用，并为 NDV 发病机制提供了有见地的信息。
