Howard Timothy R, Crow Marni S, Greco Todd M, Lum Krystal K, Li Tuo, Cristea Ileana M
Department of Molecular Biology, Princeton Universitygrid.16750.35, Princeton, New Jersey, USA.
mSystems. 2021 Jun 29;6(3):e0039721. doi: 10.1128/mSystems.00397-21. Epub 2021 Jun 22.
DNA sensors are critical components of innate immunity that enable cells to recognize infection by pathogens with DNA genomes. The interferon-inducible protein X (IFIX), a member of the PYHIN protein family, is a DNA sensor capable of promoting immune signaling after binding to double-stranded DNA (dsDNA) within either the nucleus or cytoplasm. Here, we investigate the impact of IFIX on the cellular proteome upon introduction of foreign DNA to the nucleus or the cytoplasm as well as regulatory hubs that control IFIX subcellular localization. Using quantitative mass spectrometry, we define the effect of CRISPR-mediated IFIX knockout on nuclear and cytoplasmic proteomes in fibroblasts. Proteomes are probed in response to either nuclear viral DNA, during herpes simplex virus 1 (HSV-1) infection, or cytoplasmic viral DNA, following transfection with dsDNA derived from vaccinia virus (VACV 70-mer). We show that IFIX broadly impacts nuclear and cytoplasmic proteomes, inducing alterations in the abundances of immune signaling, DNA damage response, and vesicle-mediated transport proteins. To characterize IFIX properties that regulate its localization during DNA sensing, we perform deletion and mutagenesis assays. We find that IFIX contains a multipartite nuclear localization signal (NLS) and highlight the main contributing motif for its nuclear localization. Using immunoaffinity purification, we identify IFIX acetylation and phosphorylation sites. Mutations to acetyl or charge mimics demonstrate that K138 acetylation, positioned within the NLS, affects nuclear localization. Altogether, our study establishes a mechanism regulating IFIX subcellular localization and contextualizes this localization with the involvement of IFIX in host cell responses to pathogenic DNA. Mammalian cells must be able to detect and respond to invading pathogens to prevent the spread of infection. DNA sensors, such as IFIX, are proteins that bind to pathogen-derived double-stranded DNA and induce antiviral cytokine expression. Here, we characterize the host proteome changes that require IFIX during both viral infection and DNA transfection. We show IFIX mobilizes numerous pathways and proteome alterations within the nucleus and the cytoplasm, pointing to a multifunctional protein with roles in immune signaling, DNA damage response, and transcriptional regulation. We next interrogate the IFIX domains required for nuclear localization, discovering its regulation via a multipartite nuclear localization motif. The acetylation of this motif promotes IFIX cytoplasmic localization, in agreement with its detection of pathogenic DNA in both the nucleus and the cytoplasm. This study established NLS acetylation as a conserved mechanism for regulating the localization of nuclear DNA sensors from the PYHIN family of proteins.
DNA传感器是先天免疫的关键组成部分，可使细胞识别具有DNA基因组的病原体感染。干扰素诱导蛋白X（IFIX）是PYHIN蛋白家族的成员，是一种DNA传感器，在与细胞核或细胞质内的双链DNA（dsDNA）结合后能够促进免疫信号传导。在这里，我们研究了将外源DNA引入细胞核或细胞质后IFIX对细胞蛋白质组的影响，以及控制IFIX亚细胞定位的调控枢纽。使用定量质谱，我们确定了CRISPR介导的IFIX敲除对成纤维细胞核和细胞质蛋白质组的影响。在单纯疱疹病毒1（HSV-1）感染期间，针对核病毒DNA或在用痘苗病毒衍生的dsDNA（VACV 70-mer）转染后针对细胞质病毒DNA探测蛋白质组。我们表明，IFIX广泛影响核和细胞质蛋白质组，诱导免疫信号传导、DNA损伤反应和囊泡介导的转运蛋白丰度的改变。为了表征在DNA传感过程中调节其定位的IFIX特性，我们进行了缺失和诱变分析。我们发现IFIX包含一个多部分核定位信号（NLS），并突出了其核定位的主要贡献基序。使用免疫亲和纯化，我们鉴定了IFIX的乙酰化和磷酸化位点。乙酰化或电荷模拟突变表明，位于NLS内的K138乙酰化影响核定位。总之，我们的研究建立了一种调节IFIX亚细胞定位的机制，并将这种定位与IFIX参与宿主细胞对致病性DNA的反应联系起来。哺乳动物细胞必须能够检测并应对入侵的病原体，以防止感染扩散。DNA传感器，如IFIX，是与病原体衍生的双链DNA结合并诱导抗病毒细胞因子表达的蛋白质。在这里，我们表征了病毒感染和DNA转染过程中需要IFIX的宿主蛋白质组变化。我们表明，IFIX在细胞核和细胞质内动员了众多途径和蛋白质组改变，表明它是一种在免疫信号传导、DNA损伤反应和转录调控中起作用的多功能蛋白质。接下来，我们研究了核定位所需的IFIX结构域，通过多部分核定位基序发现了其调控机制。该基序的乙酰化促进IFIX的细胞质定位，这与其在细胞核和细胞质中检测致病性DNA一致。这项研究确立了NLS乙酰化作为调节PYHIN蛋白家族中核DNA传感器定位的保守机制。
