Ubiquitin E3 ligase SPOP is a host negative regulator of enterovirus 71-encoded 2A protease.

IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.

Methotrexate and Triptolide regulate Notch signaling pathway by targeting the Nedd4-Numb axis.

Methotrexate (MTX) is used to treat rheumatoid arthritis, acute leukemia, and psoriasis. MTX can cause certain side effects, such as myelosuppression, while the exact mechanism of myelosuppression caused by MTX is unknown. Notch signaling pathway has been considered to be essential to regulate hematopoietic stem cell (HSC) regeneration and homeostasis, thus contributing to bone marrow hematopoiesis. However, whether MTX affects Notch signaling remains unexplored. Here, our study provides evidence that MTX strongly suppresses the Notch signaling pathway. We found that MTX inhibited the interaction between Nedd4 with Numb, thus restricting K48-linked polyubiquitination of Numb and stabilizing Numb proteins. This in turn inhibited the Notch signaling pathway by reducing Notch1 protein levels. Interestingly, we found that a monomeric drug, Triptolide, is capable of alleviating the inhibitory effect of MTX on Notch signaling pathway. This study promotes our understanding of MTX-mediated regulation of Notch signaling and could provide ideas to alleviate MTX-induced myelosuppression.

The OTUD1-Notch2-ICD axis orchestrates allogeneic T cell-mediated graft-versus-host disease.

Disorders of the ubiquitin-proteasome system (UPS) are known to influence the incidence and mortality of various diseases. It remains largely unknown whether and how the UPS affects the onset and progression of acute graft-verus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study demonstrated that the deubiquitinase OTUD1 is an essential regulator of aGVHD. Activation of CD4+ T cells after allo-HSCT, elevated the protein levels of OTUD1, which in turn interacted with the Notch2-ICD (NICD) to cleave the ubiquitin of NICD at the K1770 site, thereby inducing NICD protein accumulations in T cells. OTUD1-driven NICD signaling promoted the differentiation and functions of Th1 and Th17 cells and amplified the cascade of aGVHD. Moreover, by screening a FDA-approved drugs library the study identified dapagliflozin as an inhibitor targeting the OTUD1/NICD axis. Dapagliflozin administration significantly prolonged the survival of aGVHD mice. This study characterized a previously unknown role of OTUD1 in T cell-mediated allogeneic responses and provided a promising therapeutic strategy to target OTUD1 for the alleviation of aGVHD.

HSV-1-encoded ICP0 degrades the host deubiquitinase BRCC36 to antagonize interferon antiviral response.

Type I interferon (IFN-I) plays pivotal roles in defense against viral infection. HSV-1 has evolved multiple strategies to evade IFN-I antiviral response. In this study, we revealed a new mechanism that HSV-1-encoded ICP0 regulates the host deubiquitinase BRCC36 to inhibit IFN-I antiviral response. We found that HSV-1 infection rapidly downregulates BRCC36 proteins at the early stage of infection. Further studies demonstrated that HSV-1-encoded ICP0 induces K48-linked polyubiquitination and degradation of BRCC36. Importantly, HSV-1-induced BRCC36 degradation promotes downmodulation of IFN-I receptor IFNAR1, thus restricting host IFN-I antiviral response to facilitate HSV-1 early infection. These findings uncover a novel immune evasion mechanism exploited by HSV-1 and could provide potential strategies for anti-HSV-1 therapy.

BRCC36 functions noncatalytically to promote antiviral response by maintaining STAT1 protein stability.

Viral infection is a serious threat to both normal population and clinical patients. STAT1 plays central roles in host defense against viral infection. How STAT1 protein maintains stable in different conditions remains largely unknown. Here, we identified BRCC36 as a potent regulator of STAT1 protein stability. Mechanistically, BRCC36 maintains STAT1 levels by utilizing USP13 to form a balanced complex for antagonizing Smurf1-mediated degradation. Importantly, cellular BRCC36 deficiency results in rapid downregulation of STAT1 during viral infection, whereas a supplement of BRCC36 maintains STAT1 protein levels and host antiviral immunity in vivo. Moreover, we revealed that BRCC36 expression was downregulated in allogeneic HSC transplantation (allo-HSCT) mice that showed increased susceptibility to viral infection. Supplementing BRCC36 enhanced antiviral response of allo-HSCT mice by maintaining STAT1 stability. This study uncovers a critical role of BRCC36 in STAT1 protein stability and could provide potential strategies for enhancing clinical antiviral therapy.

Targeting UBE4A Revives Viperin Protein in Epithelium to Enhance Host Antiviral Defense.

Mutation and prevalence of pathogenic viruses prompt the development of broad-spectrum antiviral strategies. Viperin is a potent antiviral protein that inhibits a broad range of viruses. Unexpectedly, we found that Viperin protein production in epithelium is defective in response to both viruses and interferons (IFNs). We further revealed that viruses and IFNs stimulate expression of the acetyltransferase HAT1, which induces Lys197-acetylation on Viperin. Viperin acetylation in turn recruits UBE4A that stimulates K6-linked polyubiquitination at Lys206 of Viperin, leading to Viperin protein degradation. Importantly, UBE4A deficiency restores Viperin protein production in epithelium. We then designed interfering peptides (IPs) to inhibit UBE4A binding with Viperin. We found that VIP-IP3 rescues Viperin protein production in epithelium and therefore enhances cellular antiviral activity. VIP-IP3 renders mice more resistant to viral infection. These findings could provide strategies for both enhancing host broad-spectrum antiviral response and improving the efficacy of IFN-based antiviral therapy.

ADP-ribosyltransferase PARP11 modulates the interferon antiviral response by mono-ADP-ribosylating the ubiquitin E3 ligase ß-TrCP.

Outbreaks of viral infections are a global health burden. Although type I interferon (IFN-I) exerts broad-spectrum antiviral effects, its antiviral efficacy in host cells is largely restricted by viruses. How the antiviral efficacy of IFN-I can be improved remains to be explored. Here, we identified the ADP-ribosyltransferase poly(ADP-ribose) polymerase family member 11 (PARP11) as a potent regulator of IFN-I antiviral efficacy. PARP11 does not restrict IFN-I production induced by vesicular stomatitis virus or Sendai virus but inhibits the strength of IFN-I-activated signalling. Mechanistically, PARP11 mono-ADP-ribosylates the ubiquitin E3 ligase ß-transducin repeat-containing protein (ß-TrCP). Mono-ADP-ribosylation of ß-TrCP promotes IFNα/ß receptor subunit 1 (IFNAR1) ubiquitination and degradation. Moreover, PARP11 expression is upregulated by virus infections, including vesicular stomatitis virus, herpes simplex virus-1 and influenza A virus, thus promoting ADP-ribosylation-mediated viral evasion. We further highlight the potential for repurposing clinical ADP-ribosylation inhibitors. We found that rucaparib can target PARP11 to stabilize IFNAR1 and therefore exhibits efficient enhancement of IFN-I signalling and the host antiviral response. Consequently, rucaparib renders mice more resistant to viral infection. Our study updates the understanding of how ß-TrCP regulates its substrates and may provide a druggable target for improving IFN antiviral efficacy.

Ubiquitin C-terminal hydrolase-L3 promotes interferon antiviral activity by stabilizing type I-interferon receptor.

Type-I interferons (IFN-I) are important antiviral drugs which are widely used in clinical therapy of diverse viral infections. However, understanding the detailed mechanisms for IFN-I antiviral signaling remains a major challenge, and may provide novel targets for IFN-based antiviral therapy. So far, the roles of deubiquitinases (DUBs) in regulating IFN-I antiviral activity are still largely unexplored. Here, we find that Ubiquitin C-terminal hydrolase-L3 (UCHL3) plays an important role in regulating type I-interferon (IFN-I) mediated antiviral response. Interestingly, we find that UCHL3 regulates COPS5-dependent deneddylation of Cullin1, which is an essential component of SCFß-TrCP complex and associated with SCFß-TrCP activities. Furthermore, we reveal that UCHL3 physically interacts with COPS5, and determines the level and protein stability of cellular COPS5 by deubiquitinating COPS5. We further demonstrate that UCHL3 upregulates the levels of SCFß-TrCP substrates including IFN-I receptor IFNAR1, which enhances IFN-I mediated signaling pathway and antiviral activity. These findings identify COPS5 as a novel in vivo substrate of UCHL3, and uncover the deubiquitination-deneddylation mediated regulation for IFN-I signaling and antiviral function, which may provide a novel strategy for improving IFN-based antiviral therapy.

Deubiquitinase USP33 is negatively regulated by ß-TrCP through ubiquitin-dependent proteolysis.

Ubiquitin-mediated proteolysis regulates cellular levels of various proteins, and therefore plays important roles in controlling cell signaling and disease progression. The Skp1-Cul1-F-box ubiquitin ligase ß-TrCP is recognized as an important negative regulator for numerous key signaling proteins. Recently, the deubiquitinases (DUBs) have turned out to be essential to regulate signaling pathways related to human diseases. However, whether ß-TrCP is able to regulate the deubiquitinase family members remains largely unexplored. Here, we found that ß-TrCP downregulated cellular levels of endogenous USP33. We also revealed that ß-TrCP interacted with USP33 independently of the classic binding motif for ß-TrCP, and mediated USP33 degradation via the ubiquitin proteasome pathway. Furthermore, we found that the WD40 motif of ß-TrCP and 201-400 amino acid motif of USP33 are required for the interaction between ß-TrCP and USP33. Consequently, ß-TrCP attenuated USP33-mediated inhibition of cell proliferation and cell invasion. Taken together, our study clarified that the E3 ligase ß-TrCP regulates cellular USP33 levels by the ubiquitin-proteasomal proteolysis.

Ubiquitin-dependent Turnover of Adenosine Deaminase Acting on RNA 1 (ADAR1) Is Required for Efficient Antiviral Activity of Type I Interferon.

Adenosine deaminase acting on RNA 1 (ADAR1) catalyzes RNA editing of cellular and viral RNAs. Besides RNA editing, ADAR1 has recently been shown to play important roles in maintaining the body balance, including tissue homoeostasis, organ development, and autoimmune regulations, by inhibiting both IFN production and subsequent IFN-activated pathways. Accordingly, the question was raised how IFN signaling induced by viral infections overcomes the inhibitory effect of constitutively expressed ADAR1 (ADAR1-P110) to execute efficient antiviral activity. Here we unexpectedly found that IFN signaling promoted Lys48-linked ubiquitination and degradation of ADAR1-P110. Furthermore, we identified the E3 ligase ß transducin repeat-containing protein responsible for IFN-mediated ADAR1-P110 down-regulation. IFN signaling promoted the interaction between ß transducin repeat-containing protein and ADAR1-P110 as well as protein turnover of ADAR1-P110. Moreover, we found that both lysine 574 and 576 are essential for ADAR1-P110 ubiquitination. Critically, we demonstrated that down-regulation of ADAR1-P110 is required for IFN signaling to execute efficient antiviral activity during viral infections. These findings renew the understanding of the mechanisms by which IFN signaling acts to achieve antiviral functions and may provide potential targets for IFN-based antiviral therapy.

Smurf1 represses TNF-α production through ubiquitination and destabilization of USP5.

Ubiquitin-specific peptidase 5 (USP5) has been demonstrated to be critical for the production of Tumor Necrosis Factor-alpha (TNF-α), a pivotal mediator for inflammatory responses. Besides, USP5 regulates p53 activation and DNA repair. However, the mechanism underlying the regulation of USP5, especially its responsible E3 ligase is still unclear. Here we found that Smad ubiquitination regulatory factor 1 (Smurf1) down regulated protein expression of USP5, and the E3 enzyme activity of Smurf1 was required for this function. We also revealed that Smurf1 interacted with USP5 and mediated its degradation via the ubiquitin proteasome pathway. Consequently, Smurf1 inhibited the production of TNF-α through down-regulation of USP5. Taken together, our study for the first time clarified that the E3 ligase Smurf1 regulates USP5 protein stability and USP5-mediated TNF-α production through the ubiquitin proteasome pathway.