Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan.
Institute of Biochemistry and Molecular Biology, National Taiwan University Medical College, Taipei, Taiwan.
J Biomed Sci. 2018 Apr 11;25(1):34. doi: 10.1186/s12929-018-0434-4.
SP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-κB (NF-κB) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions.
We examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions.
Using reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-κB-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1-276) of SP110 with p50, a subunit of NF-κB, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-α (TNFα) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions.
The findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.
干扰素诱导核蛋白 SP110 属于 SP100/SP140 蛋白家族。最近，我们发现 SP110 异构体 SP110b 通过调节核因子-κB（NF-κB）活性来控制宿主对结核分枝杆菌感染的固有免疫。然而，SP110 的结构与其细胞功能之间的关系尚不清楚。在这项研究中，我们提供了实验数据，说明了负责其功能的蛋白质结构域。
我们通过荧光素酶报告基因检测分析了 SP110 异构体和一系列 SP110 缺失突变体对转录调控的影响。我们还采用共聚焦显微镜确定了增强型绿色荧光蛋白标记的 SP110 异构体和 SP110 突变体的细胞分布。此外，我们进行了免疫沉淀和 Western 印迹分析，以确定 SP110 负责蛋白相互作用的区域。
通过报告基因检测，我们首先证明 SP110 异构体对 NF-κB 介导的转录具有不同的调节作用，支持 SP110 异构体可能具有不同的细胞功能的观点。SP110 缺失突变体的分析表明，SP110 的 N 端片段（氨基酸 1-276）与细胞质中 NF-κB 的 p50 亚基相互作用，在核内对 p50 驱动的肿瘤坏死因子-α（TNFα）启动子活性的下调起着至关重要的作用，而 SP110 的中间和 C 端区域将其定位于各种细胞区室。令人惊讶的是，在 SP110 的中间区域鉴定出一个核仁定位信号（NoLS），它包含一个单部分核定位信号（NLS）和一个双部分 NLS。SP110 中隐藏的 NoLS 的鉴定表明，尽管该蛋白在核质中形成核斑点，但它可能被引导到核仁中，以在某些细胞条件下执行独特的功能。
本研究阐明了 SP110 的多结构域结构，不仅确定了 SP110 转录调控、细胞易位和蛋白相互作用所需的功能结构域，还暗示 SP110 通过其在核仁中的意外活性具有其他功能。
