The Molecular Mechanisms Of USP25Regulation Ⅰ Interferon Signaling Pathway

Virus invading organisms can initiate the immune system of host, which mainly includes non-specific natural immune defensive system and specific immune responsive defensive system. Innate immunity response is usually induced in the early period of pathogen infecting. Interferons (IFNs) as important antiviral factors, can clear the virus. Viral infections triggered a series of signaling events that lead to induction of type I IFNs. Type I IFNs then activate the JAK-STAT signal transduction pathways, leading to transcriptional induction of a wide range of downstream antiviral genes and subsequent innate antiviral response.Ubiquitination is a reversible process. Deubiquitination mediated by deubiquitination enzymes (DUBs) which also plays an important role in the innate immune response. Deubiquitination enzymes have been widespread concern in recent years, especially the ubiquitin-specific proteases (USPs) which have diversity and complexity of the structure and function. The recent reports which reveal a wide range of biological activity on the USPs is endless. In order to more fully understand the structural and functional characteristics of this family, we have a more in-depth study around the structure and function of the ubiquitin-specific protease USP25as well as its role in the regulation of the interferon signaling pathway. The main research works were as following:1. USP25negatively regulates SEV-induced activation of ISRE and the expression of ISGsIFN-stimulated response element (ISRE), as a response element of the interferon stimulated genes (ISGs) promoter regulatory region, can start the transcription of ISGs and plays a central role in the antiviral innate immune response. We screened a pool of iRNAs targeting54human USPs genes for their abilities to regulate SEV-induced activation of the ISRE promoter in reporter assays and found that USP25significantly potentiated SEV-induced activation of the ISRE promoter. Further overexpression of USP25strongly inhibited SEV-induced activation of ISRE promoter in a dose-dependent manner. Real-time RT-PCR analysis suggested that the mRNA levels of multiple ISGs were markedly reduced after stimulation. These results suggest that USP25indeed negatively regulates antiviral innate immune response.2. The molecular mechanisms of USP25negatively regulation type ⅠIFN signaling pathwayReporter assays and Real-Time RT-PCR indicated that USP25significantly inhibited the transcription of SEV-induced interferon-β(IFN-β). Further reporter assays show that knockdown of USP25potentiated SeV-induced activation of the IFN-β promoter. Collectively, the findings suggesting that USP25negatively regulated type I IFNs signaling pathways.Both transcription factors interferon regulatory factor-3(IRF3) and nuclear factor κB (NF-κB) have a pivotal role in coordinating the virus-inducible activation of type I IFNs. Our reporter assays suggest that USP25negatively inhibited activation of IRF-3and NF-κB as well as their phosphorylation which indicated that USP25negatively regulates IFN-p expression by inhibiting SEV-induced activation of IRF-3and NF-κB.3. Identified the deubiquitination activity and deubiquitination activity sites of USP25Although USP25belongs to a member of DUBs, it is unlikely that all predicted DUBs are truly specific for Ub. Both purified USP25protein and in vivo ectopically expressed USP25cleave both K48-and K63-linked poly-ubiquitin chains to provide free ubiquitin.The catalytic domain of USPs contains two short and well-conserved motifs, called Cys and His boxes, which make up the catalytic triad together with Asp/Asn.178C (lysine) and607H (histidine) are deubiquitination active sites of USP25by conserved sequence analysis. To identify whether this speculation is right, we constructed the expression plasmids for the mutant of USP25. The results suggest that178C (lysine) and607H (histidine) are deubiquitination active sites of USP25by Western blot.4. The deubiquitinating activity of USP25were required for suppressing SEV-induced IFN signalingReporter assays indicated that the catalytic USP25mutants devoid of DUB activity significantly lost the ability of USP25WT-mediated IFN inhibition. These results indicate that the DUB activity of USP25is involved in the inhibition of type I IFNs induction. The deubiquitinating activity of USP25was required for suppressing SEV-induced IFN signaling. Meanwhile USP25inhibited RIG-I-, IPS-1-, TRAF2-, and TRAF6-mediated activation of the IFN-P promoter. Endogenous coimmunoprecipitation experiments indicated that USP25deubiquitinate RIG-I, TRAF2and TRAF6, but the mutant of USP25 which lost deubiquitinating activity can not deubiquitinate RIG-I, TRAF2and TRAF6. The results suggest that USP25play an important role in virus-induced IFN signaling.