Negative regulation of the RLR‐mediated IFN signaling pathway by duck ubiquitin‐specific protease 18 (USP18)

Abstract

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 5'UTR, a 1,131-bp open reading frame and a 356-bp 3'UTR, 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-κB (NF-κB) and interferon regulatory factor 1 (IRF1) activity, and reduced IFN-β production following 5' triphosphate double-stranded RNA (5'ppp dsRNA) or lipopolysaccharide (LPS) stimulation. duUSP18 knockdown significantly activated 5'ppp dsRNA-induced and LPS-induced NF-κB and IRF1 activation, and induced IFN-β 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 5'ppp 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-β 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.

β-hydroxybutyrate Impedes the Progression of Alzheimer’s Disease and Atherosclerosis in ApoE-Deficient Mice

“…Based on the recent evidence that identified ApoE4-specific differentially expressed ChP genes [19], we next examined further in ECPC4 cells whether β-OHB regulates the expressions of ApoE4-specific differentially expressed ChP genes, ubiquitin specific peptidase 18 (usp18), IFN-induced protein with tetratricopeptide repeats 3 and 1 (ifit3, ifit1), interferon, alpha-inducible protein 27-like 2A (ifi27l2a), interferon-induced protein 44 (ifi44), guanylate-binding protein 3 (gbp3), IFN-regulatory factor 7 (irf7), and receptor transporter protein 4 (rtp4), the biological functions of which include regulation of autoimmunity by macrophages and dendritic cells, and maintenance of blood-brain barrier integrity [20][21][22]. We observed that, except ifi27l2a, expressions of usp18, ifit3, ifit1, ifi44, gbp3, irf7, and rtp4 were induced by oxLDL treatment, and β-OHB treatment reversed oxLDL-stimulated expressions of usp18, ifit3, ifit1, ifi44, gbp3, irf7, and rtp4 (Figure 3).…”

Versatility of USP18 in physiology and pathophysiology

“…it cleaves ubiquitin or Ubls from their target molecules . Initially, USP18 was cloned from mice expressing leukemia-fusion protein AML1-ETO and, afterwards, from virus-infected porcine alveolar macrophages and human melanoma cell lines Gu et al, 2019).…”

“…STATs activate the interferon-stimulated response element (ISRE) in the nucleus which, in turn, starts antiviral gene transcription. USP18 binds with IFNAR2, one of the two IFNAR subunits, and prevents JAK1 phosphorylation Basters et al, 2017;Gu et al, 2019). Apart from IFNs, other factors, such as TNF-α or LPS, can also boost USP18 expression, reduce antiviral defence and facilitate opportunistic viral infections.…”

“…Ubiquitin-specific peptidase 18 (USP18) is a cysteine protease of a dual nature. As an enzyme, it cleaves ubiquitin-like molecules (Ubls) from their target proteins, while as a substrate for an interferon (IFN) receptor it acts as an IFN type I and III signalling suppressor (Hoeller et al, 2006;Manini at al., 2013;Hong et al, 2014;Ketscher & Knobeloch, 2015;Honke et al, 2016;Mac-Parland et al, 2016;An et al, 2017;Basters et al, 2017;Mustachio et al, 2017;Basters et al, 2018;Shaabani et al, 2018;Gu et al, 2019). These diverse functions make USP18 an interesting object for research and many studies describing this unique molecule have been published recently.…”

“…These diverse functions make USP18 an interesting object for research and many studies describing this unique molecule have been published recently. Among others, its contribution to cell signalling, response to viral and bacterial infections, development of several malignancies and development of autoimmune diseases have been described (Liu et al, 1999;Ritchie et al, 2004;Hoeller et al, 2006;Catic et al, 2007;Duex & Sorkin, 2009;François-Newton et al, 2011;Murray et al, 2011;Burkart et al, 2012;Guo et al, 2012;Yim et al, 2012;Coit et al, 2013;Honke et al, 2013;Hong et al, 2014;Zhang et al, 2015;Honke et al, 2016;Ying et al, 2016;An et al, 2017;Arimoto et al, 2017;Basters et al, 2017;Mustachio et al, 2017;Basters et al, 2018;Shaabani et al, 2018;Gu et al, 2019). USP18 was also found to serve as an inhibitor of cardiac remodelling (Ying et al, 2016).…”