DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1.

DEAD-box helicase (DDX) family members play differential roles in regulating innate antiviral immune response. However, the physiological roles played by DDX4 in antiviral innate immunity remain unclear. In this study, we unveiled that DDX4 acts as a positive regulatory molecule of Type-I interferon (IFN-I)-mediated antiviral activity. Our findings demonstrate that IFN-I upregulates DDX4 protein levels, and subsequently, overexpression of DDX4 enhances the IFN-I-mediated signaling pathway. This creates a positive feedback loop that amplifies the antiviral response. DDX4 was found to bind with deubiquitinase ubiquitin-specific protease 7 (USP7), leading to the disruption of the interaction between USP7 and suppressor of cytokine signaling 1 (SOCS1) and the subsequent degradation of SOCS1. This process enhances the antiviral function of IFN-I. Our findings provide new insights into the regulatory role of DDX4 in the IFN-I response.IMPORTANCEDDX4, identified as a putative RNA helicase that modulates RNA secondary structure through RNA binding, is primarily acknowledged for its role in regulating mRNA translation within the germline. Nevertheless, the extent of DDX4's involvement in the antiviral innate immune response remains largely unexplored. This study presents evidence of a previously unrecognized positive feedback loop between DDX4 and the antiviral response, suggesting that disruption of this loop may serve as a novel mechanism for viral evasion. Furthermore, our findings elucidate a positive regulatory mechanism by which the DDX4/USP7/SOCS1 axis mediates the antiviral activity of Type-I interferon, which provides new insight into strategies for improving the efficacy of IFN-based antiviral therapy.

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.

Cycloheximide (CHX) Chase Assay to Examine Protein Half-life.

Cycloheximide (CHX) is a small molecule derived from Streptomyces griseus that acts as fungicide. As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and used to observe intracellular protein degradation and to determine the half-life of a given protein in eukaryotes. Here, we present a complete experimental procedure of the CHX chase assay. Graphical overview.

Vitamin C promotes ACE2 degradation and protects against SARS-CoV-2 infection.

ACE2 is a major receptor for cellular entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2 binding, strategies to flexibly and sufficiently reduce ACE2 levels for the prevention of SARS-CoV-2 infection have not been explored. Here, we reveal vitamin C (VitC) administration as a potent strategy to prevent SARS-CoV-2 infection. VitC reduces ACE2 protein levels in a dose-dependent manner, while even a partial reduction in ACE2 levels can greatly inhibit SARS-CoV-2 infection. Further studies reveal that USP50 is a crucial regulator of ACE2 levels. VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination of ACE2 at Lys788 and subsequent degradation of ACE2 without affecting its transcriptional expression. Importantly, VitC administration reduces host ACE2 levels and greatly blocks SARS-CoV-2 infection in mice. This study reveals that ACE2 protein levels are down-regulated by an essential nutrient, VitC, thereby enhancing protection against infection of SARS-CoV-2 and its variants.

Linear ubiquitination improves NFAT1 protein stability and facilitates NFAT1 signalling in Kawasaki disease.

NFAT1 is known for its roles in T cell development and activation. So far, the phosphorylation of NFAT1 has been extensively studied, but the other post-translational modifications of NFAT1 remain largely unknown. In this study, we reported that NFAT1 is a linearly ubiquitinated substrate of linear ubiquitin chain assembly complex (LUBAC). LUBAC promoted NFAT1 linear ubiquitination, which in turn inhibited K48-linked polyubiquitination of NFAT1 and therefore increased NFAT1 protein stability. Interestingly, the linear ubiquitination levels of NFAT1 in patients with the Kawasaki disease were upregulated. Further studies demonstrated that the patients with the Kawasaki disease had increased mRNA levels of HOIL-1L. These findings revealed a linearly ubiquitinated substrate of LUBAC and an important biological function of NFAT1 linear ubiquitination in the Kawasaki disease and therefore may provide a novel strategy for the treatment of the Kawasaki disease.

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.

Menthone inhibits type-I interferon signaling by promoting Tyk2 ubiquitination to relieve local inflammation of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease. RA development is mediated by the abnormal activation of multiple signaling pathways. Recent studies have revealed that type-I interferon (IFN-I) signaling plays an essential role in the occurrence and development of RA. However, how to target IFN-I signaling to develop anti-rheumatoid arthritis drugs remains largely unexplored. Here, our study showed that IFN-I signaling was over-activated in articular synovial cells from collagen II-induced arthritis (CIA) mice. Interestingly, we found that a small molecule compound, menthone, strongly inhibited the activation of the IFN-I signaling pathway. Further studies revealed that menthone promoted K48-linked polyubiquitination of Tyk2, thus lowering the protein level and stability of Tyk2. Importantly, menthone administration in the local articulus of CIA mice significantly attenuated the local inflammation in CIA mice. This study could promote our understanding of rheumatoid arthritis, and also suggests a potential strategy to develop anti-RA drugs.

E3 ubiquitin ligase MID1 ubiquitinates and degrades type-I interferon receptor 2.

Type I interferon (IFN-I) is a common biological molecule used for the treatment of viral diseases. However, the clinical antiviral efficacy of IFN-I needs to be greatly improved. In this study, IFN-I receptor 2 (IFNAR2) was revealed to undergo degradation at the protein level in cells treated with IFN-I for long periods of time. Further studies found a physical interaction between the E3 ubiquitin ligase midline-1 (MID1) and IFNAR2. As a consequence, MID1 induced both K48- and K63-linked polyubiquitination of IFNAR2, which promoted IFNAR2 protein degradation in a lysosome-dependent manner. Conversely, knockdown of MID1 largely restricted IFN-I-induced degradation of IFNAR2. Importantly, MID1 regulated the strength of IFN-I signalling and IFN-I-induced antiviral activity. These findings reveal a regulatory mechanism of IFNAR2 ubiquitination and protein stability in IFN-I signalling, which could provide a potential target for improving the antiviral efficacy of IFN-I.

Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis.

Depression is a serious public-health issue. Recent reports have suggested higher susceptibility to viral infections in depressive patients. However, how depression affects antiviral innate immune signaling remains unknown. Here, we revealed a reduction in expression of Abelson helper integration site 1 (AHI1) in the peripheral blood mononuclear cells (PBMCs) and macrophages from the patients with major depressive disorder (MDD), which leads to attenuated antiviral immune response. We found that depression-related arginine vasopressin (AVP) induces reduction of AHI1 in macrophages. Further studies demonstrated that AHI1 is a critical stabilizer of basal type-I-interferon (IFN-I) signaling. Mechanistically, AHI1 recruits OTUD1 to deubiquitinate and stabilize Tyk2, while AHI1 reduction downregulates Tyk2 and IFN-I signaling activity in macrophages from both MDD patients and depression model mice. Interestingly, we identified a clinical analgesic meptazinol that effectively stimulates AHI1 expression, thus enhancing IFN-I antiviral defense in depression model mice. Our study promotes the understanding of the signaling mechanisms of depression-mediated antiviral immune dysfunction, and reveals meptazinol as an enhancer of antiviral innate immunity in depressive patients.

LATS1 is a central signal transmitter for achieving full type-I interferon activity.

Interferons (IFNs) have broad-spectrum antiviral activity to resist virus epidemic. However, IFN antiviral efficacy needs to be greatly improved. Here, we reveal that LATS1 is a vital signal transmitter governing full type-I IFN (IFN-I) signaling activity. LATS1 constitutively binds with the IFN-I receptor IFNAR2 and is rapidly tyro-phosphorylated by Tyk2 upon IFN-I engagement. Tyro-phosphorylation of LATS1 promotes LATS1 activation and YAP degradation, thereby promoting IFN-mediated antiproliferation activity. Moreover, activated LATS1 translocates into the nucleus and induces CDK8-Ser62 phosphorylation, which in turn phosphorylates STAT1 at Ser727 and induces full IFN-I antiviral activity. LATS1 deficiency restricts in vivo IFN-I signaling and attenuates host antiviral immune response. Our study identifies IFN-I as a previously unidentified extracellular diffusible ligand signal for activation of the Hippo core LATS1 pathway and reveals Tyk2-LATS1-CDK8 as a complete signaling cascade controlling full IFN-I activity.

A Natural Plant Ingredient, Menthone, Regulates T Cell Subtypes and Lowers Pro-inflammatory Cytokines of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with nearly 1.6 billion patients worldwide and an incidence of 0.5-1%. In recent years, basic and clinical studies have revealed that immune cell responses and corresponding secretion of inflammatory factors are important in the control of RA development. Our study found that a natural plant ingredient, menthone, could be used as a potential antirheumatism compound. In vivo observations demonstrated that menthone alleviates collagen II-induced arthritis (CIA) in mice. Furthermore, we found that menthone regulates the number of Th1 and Th17 cells in CIA mice. Importantly, menthone significantly inhibits the release of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6, in CIA mice. Our study suggests a potential component for the development of drugs to treat rheumatoid arthritis.

High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction.

High-salt diets have recently been implicated in hypertension, cardiovascular disease, and autoimmune disease. However, whether and how dietary salt affects host antiviral response remain elusive. Here, we report that high salt induces an instant reduction in host antiviral immunity, although this effect is compromised during a long-term high-salt diet. Further studies reveal that high salt stimulates the acetylation at Lys663 of p97, which promotes the recruitment of ubiquitinated proteins for proteasome-dependent degradation. p97-mediated degradation of the deubiquitinase USP33 results in a deficiency of Viperin protein expression during viral infection, which substantially attenuates host antiviral ability. Importantly, switching to a low-salt diet during viral infection significantly enhances Viperin expression and improves host antiviral ability. These findings uncover dietary salt-induced regulation of ubiquitinated cellular proteins and host antiviral immunity, and could offer insight into the daily consumption of salt-containing diets during virus epidemics.

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.

Ubiquitin E3 ligase MID1 inhibits the innate immune response by ubiquitinating IRF3.

Interferon regulatory factor 3 (IRF3) is a critical transcription factor for inducing production of type I interferons (IFN-I) and regulating host antiviral response. Although IRF3 activation during viral infection has been extensively studied, the inhibitory regulation of IRF3 remains largely unexplored. Here, we revealed that Midline-1 (MID1) is a ubiquitin E3 ligase of IRF3 that plays essential roles in regulating the production of IFN-I. We found that MID1 physically interacts with IRF3 and downregulates IRF3 protein levels. Next, we demonstrated that MID1 can induce K48-linked polyubiquitination of IRF3, thus lowing the protein stability of IRF3. Our further studies identified Lys313 as a major ubiquitin acceptor lysine of IRF3 induced by MID1. Finally, MID1-mediated ubiquitination and degradation of IRF3 restrict IFN-I production and cellular antiviral response. This study uncovers a role of MID1 in regulating innate antiviral immunity and may provide a potential target for enhancing host antiviral activity.

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.

Ubiquitin E3 Ligase c-Cbl Is a Host Negative Regulator of Nef Protein of HIV-1.

Nef is an accessory protein encoded by human immunodeficiency virus type-1 (HIV-1) and plays important roles in regulating HIV-1 infection and viral replication. Interestingly, HIV-1 Nef can promote degradation of numerous host proteins to disrupt cellular antiviral immune response. However, how HIV-1 Nef is degraded by host factors remains largely unexplored. Here, we identified c-Cbl as a host ubiquitin E3 ligase of HIV-1 Nef. We found that c-Cbl interacts with Nef and reduces protein levels of HIV-1 Nef. Further studies demonstrated that c-Cbl promoted Lys48-linked polyubiquitination of HIV-1 Nef, thus attenuating protein stability of HIV-1 Nef. Importantly, cellular c-Cbl ubiquitinated and degraded Nef proteins produced by HIV-1 NL4-3 virions, and ultimately attenuated HIV-1 virulence for infection of THP1 cells. This study reveals a ubiquitination and proteasome-dependent degradation mechanism of HIV-1 Nef protein, and could provide potential strategies for fighting against HIV-1.

Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling.

Linear ubiquitination is a critical regulator of inflammatory signaling pathways. However, linearly ubiquitinated substrates and the biological significance of linear ubiquitination is incompletely understood. Here, we show that STAT1 has linear ubiquitination at Lys511 and Lys652 residues in intact cells, which inhibits STAT1 binding to the type-I interferon receptor IFNAR2, thereby restricting STAT1 activation and resulting in type-I interferon signaling homeostasis. Linear ubiquitination of STAT1 is removed rapidly by OTULIN upon type-I interferon stimulation, which facilitates activation of interferon-STAT1 signaling. Furthermore, viruses induce HOIP expression through the NF-κB pathway, which in turn increases linear ubiquitination of STAT1 and thereby inhibits interferon antiviral response. Consequently, HOIL-1L heterozygous mice have active STAT1 signaling and enhanced responses to type-I interferons. These findings demonstrate a linear ubiquitination-mediated switch between homeostasis and activation of type-I interferon signaling, and suggest potential strategies for clinical antiviral therapy.

USP12 translocation maintains interferon antiviral efficacy by inhibiting CBP acetyltransferase activity.

CREB-binding protein (CBP) participates in numerous transcription events. However, cell-intrinsic inhibitors of CBP are poorly defined. Here, we found that cellular USP12 interacts with the HAT domain of CBP and inhibits CBP's acetyltransferase activity. Interestingly, USP12 positively regulates interferon (IFN) antiviral signaling independently of its deubiquitinase activity. Furthermore, we found that in IFN signaling USP12 translocates from the cytoplasm to the nucleus. The decrease in cytoplasmic USP12 facilitates CBP-induced acetylation and activation of IFN signaling proteins in the cytoplasm. Moreover, USP12 accumulation in the nucleus blocks CBP-induced acetylation of phosphorylated STAT1 (p-STAT1) and therefore inhibits the dephosphorylation effects of TCPTP on p-STAT1, which finally maintains nuclear p-STAT1 levels and IFN antiviral efficacy. USP12 nuclear translocation extends our understanding of the regulation of the strength of IFN antiviral signaling. Our study uncovers a cell-intrinsic regulation of CBP acetyltransferase activity and may provide potential strategies for IFN-based antiviral therapy.