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.

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.

Ubiquitination network in the type I IFN-induced antiviral signaling pathway.

Type I IFN (IFN-I) is the body's first line of defense against pathogen infection. IFN-I can induce cellular antiviral responses and therefore plays a key role in driving antiviral innate and adaptive immunity. Canonical IFN-I signaling activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which induces the expression of IFN-stimulated genes and eventually establishes a complex antiviral state in the cells. Ubiquitin is a ubiquitous cellular molecule for protein modifications, and the ubiquitination modifications of protein have been recognized as one of the key modifications that regulate protein levels and/or signaling activation. Despite great advances in understanding the ubiquitination regulation of many signaling pathways, the mechanisms by which protein ubiquitination regulates IFN-I-induced antiviral signaling have not been explored until very recently. This review details the current understanding of the regulatory network of ubiquitination that critically controls the IFN-I-induced antiviral signaling pathway from three main levels, including IFN-I receptors, IFN-I-induced cascade signals, and effector IFN-stimulated genes.

Develop a Compact RNA Base Editor by Fusing ADAR with Engineered EcCas6e.

Catalytically inactive CRISPR-Cas13 (dCas13)-based base editors can achieve the conversion of adenine-to-inosine (A-to-I) or cytidine-to-uridine (C-to-U) at the RNA level, however, the large size of dCas13 protein limits its in vivo applications. Here, a compact and efficient RNA base editor (ceRBE) is reported with high in vivo editing efficiency. The larger dCas13 protein is replaced with a 199-amino acid EcCas6e protein, derived from the Class 1 CRISPR family involved in pre-crRNA processing, and conducted optimization for toxicity and editing efficiency. The ceRBE efficiently achieves both A-to-I and C-to-U base editing with low transcriptome off-target in HEK293T cells. The efficient repair of the DMD Q1392X mutation (68.3±10.1%) is also demonstrated in a humanized mouse model of Duchenne muscular dystrophy (DMD) after AAV delivery, achieving restoration of expression for gene products. The study supports that the compact and efficient ceRBE has great potential for treating genetic diseases.

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.

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.

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.

Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

Restoration of the tumor-suppressor function to mutant p53 by Ganoderma lucidum polysaccharides in colorectal cancer cells.

Ganoderma lucidum polysaccharides (GLPs), isolated from spores, mycelia and fruiting bodies of Ganoderma lucidum, have been suggested to possess anticancer activities in a large number of basic studies. A recent survey revealed that GLP-induced inhibition of cancer cell growth was dependent on the existence of functional p53. However, the actual role of p53-mediated tumor-suppressing pathways in facilitating the anticancer effect of GLPs is still unclear. In the present study, we investigated the interaction between GLPs and mutant p53 that exists in more than half of the known types of cancers. Our results showed that GLPs reactivated mutant p53 in colorectal cancer HT29 (p53R273H) and SW480 (p53R273H&P309S) cells while applied alone or together with 5-fluorouracil (5-FU). This reactivation further induced cell growth inhibition and apoptosis. In addition, western blot assay and in vitro cell-free apoptosis assay suggested that the activation of mutant p53 was effective in both a transcriptional-dependent and -independent pathway. Altogether, our data demonstrated for the first time that GLPs show prominent anticancer activities by reactivating several types of mutant p53. Therefore, targeting mutant p53 by GLPs alongside other chemotherapeutics may be considered as a novel treatment strategy for cancer.

Biophysical study on the interaction of an anesthetic, vecuronium bromide with human serum albumin using spectroscopic and calorimetric methods.

The interactions between an anesthetic, vecuronium bromide (VB) and human serum albumin (HSA) have been investigated systematically by steady-state/time-resolved fluorescence, circular dichroism (CD), UV-vis absorption, Fourier transform infrared spectroscopy (FTIR), mass spectroscopy and differential scanning calorimetry (DSC) methods under physiological conditions. The fluorescence quenching observed is attributed to the formation of a complex between HSA and VB, and the reverse temperature effect of the fluorescence quenching has been found and discussed. Fluorescence analysis has proved that there is one classical binding site on HSA for VB with a relative weak binding constant of 1.07 × 10(4)M(-1) at 298 K. The primary binding pattern is determined by hydrogen bonding or van der Waals forces occurring in site I of HSA with ΔG°=-2.30 × 10(4)J mol(-1), ΔS°=-233 J mol(-1)K(-1) and ΔH°=-9.23 × 10(4)J mol(-1) at 298 K. VB could slightly change the secondary structure and induce unfolding of the polypeptides of protein. The DSC results provide quantitative information on the effect of VB on the stability of serum albumin. It is shown that VB can efficiently bind with HSA and be transported to the focuses needed.

MGMT Leu84Phe polymorphism contributes to cancer susceptibility: evidence from 44 case-control studies.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase is one of the few proteins to directly remove alkylating agents in the human DNA direct reversal repair pathway. A large number of case-control studies have been conducted to explore the association between MGMT Leu84Phe polymorphism and cancer risk. However, the results were not consistent. METHODS: We carried out a meta-analysis of 44 case-control studies to clarify the association between the Leu84Phe polymorphism and cancer risk. RESULTS: Overall, significant association of the T allele with cancer susceptibility was verified with meta-analysis under a recessive genetic model (P<0.001, OR=1.30, 95%CI 1.24-1.50) and TT versus CC comparison (P=0.001, OR=1.29, 95% CI 1.12-1.50). In subgroup analysis, a significant increased risk was found for lung cancer (TT versus CC, P=0.027, OR=1.67, 95% CI 1.06-2.63; recessive genetic model, P=0.32, OR=1.64, 95% CI 1.04-2.58), whereas risk of colorectal cancer was significantly low under a dominant genetic model (P=0.019, OR=0.84, 95% CI 0.72-0.97). Additionally, a significant association between TT genetic model and total cancer risk was found in the Caucasian population (TT versus CC, P=0.014, OR=1.29, 95% CI 1.05-1.59; recessive genetic model, P=0.009, OR=1.31, 95% CI 1.07-1.61), but not in the Asian population. An increased risk for lung cancer was also verified in the Caucasian population (TT versus CC, P=0.035, OR=1.62, 95% CI 1.04-2.53; recessive genetic model, P=0.048, OR=1.57, 95% CI 1.01-2.45). CONCLUSIONS: These results suggest that MGMT Leu84Phe polymorphism might contribute to the susceptibility of certain cancers.