Negative regulation of the RLR-mediated IFN signaling pathway by duck ubiquitin-specific protease 18 (USP18)
Negative regulation of the RLR-mediated IFN signaling pathway by duck ubiquitin-specific protease 18 (USP18)
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鸭泛素特异性蛋白酶 18 (USP18) 对 RLR 介导的 IFN 信号通路的负调控
DOI：
10.1002/jcp.27208
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发表时间：
2019-04-01
影响因子：
5.6
通讯作者：
Chen, Guohong
中科院分区：
文献类型：
--
作者：
Gu, Tiantian;Lu, Lu;Chen, Guohong
泛素特异性蛋白酶18（Ubiquitin-specific protease 18，USP 18）在天然免疫中调节I型干扰素（IFN）信号通路中起重要作用，对IFN的治疗效果有着至关重要的影响。虽然在阐明USP 18在哺乳动物中的功能方面取得了重大进展，但USP 18在鸭中的作用（duUSP 18）仍然知之甚少。本研究采用反转录聚合酶链反应（RT-PCR）和快速扩增互补DNA（cDNA）末端的方法克隆了白色冠毛鸭USP 18基因。结果表明，duUSP 18 cDNA含有一个52 bp的5 UTR、一个1，131 bp的开放阅读框和一个356 bp的3UTR，编码一个376个氨基酸的蛋白。多重序列比对显示duUSP 18与其他脊椎动物的USP 18具有很高的相似性。duUSP 18的过表达抑制核因子-B（NF-B）和干扰素调节因子1（IRF 1）的活性，并且在5-三磷酸双链RNA（5 ppp dsRNA）或脂多糖（LPS）刺激后减少IFN-γ的产生。duUSP 18敲低显著激活了5 ppp dsRNA诱导的和LPS诱导的NF-B和IRF 1激活，并诱导鸭胚成纤维细胞中IFN-γ的表达。此外，定量实时PCR（qRT-PCR）显示，在用5 ppp dsRNA处理后，duUSP 18的过表达或敲低可以改变与TLR介导的IFN信号通路相关的基因的表达。此外，定点突变分析显示duUSP 18的半胱氨酸66（C66）、组氨酸313（H313）和组氨酸321（H321）对抑制IFN-γ活性至关重要。综上所述，这些结果表明，鸭USP 18在针对双链RNA病毒的先天免疫应答中在TLR介导的IFN信号通路中起重要作用，并且需要进一步研究以阐明其潜在机制，这可以为鸭疾病的治疗效果提供分子见解。
Ubiquitin-specific protease 18 (USP18) plays an important role in regulating type I interferon (IFN) signaling in innate immunity, and has a crucial impact on the IFN therapeutic effect. Although significant progress has been made in elucidating USP18 function in mammals, the role of USP18 in ducks (duUSP18) remains poorly understood. In this study, we cloned the USP18 gene from white crested ducks by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA (cDNA) ends. We determined that duUSP18 cDNA contains a 52-bp 5UTR, a 1,131-bp open reading frame and a 356-bp 3UTR, and encodes a 376-amino acid protein. Multiple sequence alignments showed that duUSP18 shares high similarity with USP18 from other vertebrates. Overexpression of duUSP18 inhibited nuclear factor-B (NF-B) and interferon regulatory factor 1 (IRF1) activity, and reduced IFN- production following 5 triphosphate double-stranded RNA (5ppp dsRNA) or lipopolysaccharide (LPS) stimulation. duUSP18 knockdown significantly activated 5ppp dsRNA-induced and LPS-induced NF-B and IRF1 activation, and induced IFN- expression in duck embryo fibroblasts. Furthermore, Quantitative real-time PCR (qRT-PCR) revealed that overexpression or knockdown of duUSP18 could alter the expression of genes related to the RLR-mediated IFN signaling pathway following the treatment with 5ppp dsRNA. In addition, site-directed mutation analysis revealed that cysteine 66 (C66), histidine 313 (H313), and histidine 321 (H321) of duUSP18 were critical for inhibiting IFN- activity. Taken together, these results suggest that duck USP18 plays an important role in innate immune responses against double-stranded RNA viruses in the RLR-mediated IFN signaling pathway, and that further studies are warranted to elucidate its underlying mechanisms, which could provide molecular insights into the effect of the treatment of duck diseases.