Ceftazidime is a potential drug to inhibit cell proliferation by increasing cellular p27.

The development of new therapeutic uses for existing drugs is important for the treatment of some diseases. Cephalosporin antibiotics stand as the most extensively utilized antibiotics in clinical practice, effectively combating bacterial infections. Here, we found that the antimicrobial drug ceftazidime strongly upregulates p27 protein levels by inhibiting p27 ubiquitination. The p27 protein is a classic negative regulator of the cell cycle. Next, we demonstrated that ceftazidime can impede the cell cycle from G1 to S phase, thus inhibiting cell proliferation. Furthermore, we found that ceftazidime promotes p27 expression and inhibits cell proliferation by reducing Skp2, which is a substrate recognition component of the Skp2-Cullin-F-box (SCF) ubiquitin ligase. Moreover, ceftazidime downregulates transcriptional expression of Skp2. Importantly, we demonstrated that ceftazidime inhibited the proliferation of tumor cells in vivo. These findings reveal ceftazidime-mediated inhibition of cell proliferation through the Skp2-p27 axis, and could provide a potential strategy for anti-tumor 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.

Ceftazidime reduces cellular Skp2 to promote type-I interferon activity.

Skp2 plays multiple roles in malignant tumours. Here, we revealed that Skp2 negatively regulates type-I interferon (IFN-I)-mediated antiviral activity. We first noticed that Skp2 can promote virus infection in cells. Further studies demonstrated that Skp2 interacts with IFN-I receptor 2 (IFNAR2) and promotes K48-linked polyubiquitination of IFNAR2, which accelerates the degradation of IFNAR2 proteins. Skp2-mediated downregulation of IFNAR2 levels inhibits IFN-I signalling and IFN-I-induced antiviral activity. In addition, we uncovered for the first time that the antibiotic ceftazidime can act as a repressor of Skp2. Ceftazidime reduces cellular Skp2 levels, thus enhancing IFNAR2 stability and IFN-I antiviral activity. This study reveals a new role of Skp2 in regulating IFN-I signalling and IFN-I antiviral activity and reports the antibiotic ceftazidime as a potential repressor of Skp2.

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.

In vitro and in vivo evaluation of virus-induced innate immunity in mouse.

Innate immunity is the first line of host defense against viral infection. As one of the innate immune cell types, antigen-presenting cells play an important role in the process of antiviral immunity. This protocol describes the analysis of innate immunity induced by vesicular stomatitis virus infection of peritoneal macrophages in vitro and in vivo detection of IFN-ß production and lung injury. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021).

Serine metabolism antagonizes antiviral innate immunity by preventing ATP6V0d2-mediated YAP lysosomal degradation.

Serine metabolism promotes tumor oncogenesis and regulates immune cell functions, but whether it also contributes to antiviral innate immunity is unknown. Here, we demonstrate that virus-infected macrophages display decreased expression of serine synthesis pathway (SSP) enzymes. Suppressing the SSP key enzyme phosphoglycerate dehydrogenase (PHGDH) by genetic approaches or by treatment with the pharmaceutical inhibitor CBR-5884 and by exogenous serine restriction enhanced IFN-ß-mediated antiviral innate immunity in vitro and in vivo. Mechanistic experiments showed that virus infection or serine metabolism deficiency increased the expression of the V-ATPase subunit ATP6V0d2 by inhibiting S-adenosyl methionine-dependent H3K27me3 occupancy at the promoter. ATP6V0d2 promoted YAP lysosomal degradation to relieve YAP-mediated blockade of the TBK1-IRF3 axis and, thus, enhance IFN-ß production. These findings implicate critical functions of PHGDH and the key immunometabolite serine in blunting antiviral innate immunity and also suggest manipulation of serine metabolism as a therapeutic strategy against virus infection.

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.

A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. RESULTS: CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. CONCLUSIONS: These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

[The enhancement of DNA binding ability of a mutated E2 (A338V) protein of HPV-2].

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.

Encystation--survival of Blastocystis hominis in immunocompetent mice abdomen cavity.

Human Blastocystis hominis were isolated from diarrhea patients' feces and cultured in vitro. Then the cultures were inoculated intraperitoneally to laboratory mice. The B. hominis in living mice were collected and inoculated again to healthy mice. The B. hominis showed dose-dependent pathogenicity in the primary inoculation. No pathogenicity was observed in the secondary inoculation. The protozoan existed in the living mice abdomen cavity for more than 6 months and the cyst was the only form. These results showed that encystation enable the parasite to avoid the immune attack in competent host and simultaneously decrease the pathogenicity to host. Intraperitoneal inoculation to laboratory mice is a good method to maintain and propagate B. hominis. This is also a good model to study the interaction of B. hominis and immune system.

Molecular epidemiological study on prevalence of human papillomaviruses in patients with common warts in Beijing area.

OBJECTIVE: To study the circulation, distribution, and genomic diversity of HPVs in common warts in Beijing area of China. METHODS: Forty eight patients with pathologically diagnosed common warts were screened for the presence of HPV with HPV type-specific PCR and direct sequencing analysis. The genomic diversity of HPVs prevalent in Chinese patients was analyzed based on LCR. RESULTS: Forty one (85.5%) samples were positive for HPV DNA, 13 (31.7%)--HPV-57, 12 (29.3%)--HPV-1a, 7 (17%)--HPV-27 and 5(12.2%)--HPV-2a. Four cases were infected with two different HPV types, two (4.9%) with HPV-1a and HPV-27, one (2.4%) with HPV-1 and HPV-57 and one (2.4%) with HPV-27 and HPV-57. In contrast to the prevalence of single strain of novel HPV-57 variant and HPV-1 prototype, two HPV-2 and three HPV-27 novel variants were found to circulate in Beijing. CONCLUSION: HPV-1, -2, -27 and -57 are predominantly prevalent in patients with common warts in Beijing.

Tumor necrosis factor-α of Red nucleus involved in the development of neuropathic allodynia.

The pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with the generation of inflammatory and neuropathic pain. The current study aims to investigate the expression of TNF-α in the brain of rats with spared nerve injury (SNI), a neuropathic pain model with the lesion of common peroneal and tibial nerves. Two weeks following SNI, the immunohistochemical results identified that the expression level of TNF-α in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the roles of TNF-α in the development of neuropathic pain, different doses of anti-TNF-α antibody (20, 2.0 and 0.2 µg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The results showed that the 50% paw withdrawal threshold (von Frey test) of SNI rats were increased by 20 and 2.0 µg/ml anti-TNF-α antibody as compared with that of the basic value and control groups (P<0.05), the analgesic effect lasted for 50 and 30 min, respectively. However, no significant analgesic effect was observed after 0.2 µg/ml antibody was microinjected into the RN. These results suggest that the TNF-α of RN is involved in the development of neuropathic allodynia in SNI rats.

[Transcriptional repressive activity of mutated E2 protein of human papillomavirus 2 (HPV-2) variant].

Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart.