Genome-wide siRNA screen for mediators of NF-κB activation
Genome-wide siRNA screen for mediators of NF-κB activation
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DOI：
10.1073/pnas.1120542109
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发表时间：
2012-02-14
影响因子：
11.1
通讯作者：
Kieff, Elliott
中科院分区：
文献类型：
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作者：
Gewurz, Benjamin E.;Towfic, Fadi;Kieff, Elliott
尽管典型的NF κ B通常对细胞增殖、存活或分化至关重要，但NF κ B过度活化可引起恶性、炎性或自身免疫性疾病。尽管进行了深入的研究，但哺乳动物NF κ B B途径功能丧失RNAi分析仅限于特定的蛋白质类别。因此，我们进行了人类全基因组siRNA筛选新的NF κ B活化途径的组成部分。使用Epstein巴尔病毒潜伏膜蛋白（LMP 1）突变体，其转录效应是典型的NF κ B依赖性，我们确定了155个蛋白质显着和实质性的重要NF κ B在HEK 293细胞中的激活。这些蛋白质包括许多以前不知道对NF κ B活化重要的激酶、磷酸酶、泛素连接酶和去泛素化酶。通过发现155个LMP 1 NF-κ B活化途径组分中的118个对于IL-1 β-和79个对于相同细胞中TNF α介导的NF-κ B活化同样重要，扩展了与其他经典NF-κ B B途径的相关性。MAP 3 K8、PIM 3和其他6种酶与LMP 1介导的NF κ B活化独特相关。大多数新的途径组分在I κ B激酶复合物（IKK）激活的上游起作用。对于所有测试的mRNA或蛋白质，证实了稳健的siRNA敲低效应。尽管多种ZC 3 H家族蛋白负调控NF κ B B，但ZC 3 H13和ZC 3 H18是活化途径组分。ZC 3 H13对LMP 1、TNF α和IL-1 β NF κ B依赖性转录至关重要，但对IKK活化无关，而ZC 3 H18对IKK活化至关重要。还鉴定了LMP 1介导的NF κ B活化的下调剂。这些实验鉴定了抑制或刺激LMP 1、IL-1 β或TNF α介导的典型NF κ B活化的多个靶点。
Although canonical NF kappa B is frequently critical for cell proliferation, survival, or differentiation, NF kappa B hyperactivation can cause malignant, inflammatory, or autoimmune disorders. Despite intensive study, mammalian NF kappa B pathway loss-of-function RNAi analyses have been limited to specific protein classes. We therefore undertook a human genome-wide siRNA screen for novel NF kappa B activation pathway components. Using an Epstein Barr virus latent membrane protein (LMP1) mutant, the transcriptional effects of which are canonical NF kappa B-dependent, we identified 155 proteins significantly and substantially important for NF kappa B activation in HEK293 cells. These proteins included many kinases, phosphatases, ubiquitin ligases, and deubiquinating enzymes not previously known to be important for NF kappa B activation. Relevance to other canonical NF kappa B pathways was extended by finding that 118 of the 155 LMP1 NF-kappa B activation pathway components were similarly important for IL-1 beta-, and 79 for TNF alpha-mediated NF kappa B activation in the same cells. MAP3K8, PIM3, and six other enzymes were uniquely relevant to LMP1-mediated NF kappa B activation. Most novel pathway components functioned upstream of I kappa B kinase complex (IKK) activation. Robust siRNA knockdown effects were confirmed for all mRNAs or proteins tested. Although multiple ZC3H-family proteins negatively regulate NF kappa B, ZC3H13 and ZC3H18 were activation pathway components. ZC3H13 was critical for LMP1, TNF alpha, and IL-1 beta NF kappa B-dependent transcription, but not for IKK activation, whereas ZC3H18 was critical for IKK activation. Down-modulators of LMP1 mediated NF kappa B activation were also identified. These experiments identify multiple targets to inhibit or stimulate LMP1-, IL-1 beta-, or TNF alpha-mediated canonical NF kappa B activation.