Whole-Cell Biotransformation of Penicillin G by a Three-Enzyme Co-expression System with Engineered Deacetoxycephalosporin†C Synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the transformation of penicillin G to phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA) for which it depends on 2-oxoglutarate (2OG) as co-substrate. However, the low activity of DAOCS and the expense of 2OG restricts its practical applications in the production of G-7-ADCA. Herein, a rational design campaign was performed on a DAOCS from Streptomyces clavuligerus (scDAOCS) in the quest to construct novel expandases. The resulting mutants showed 25∼58 % increase in activity compared to the template. The dominant DAOCS variants were then embedded into a three-enzyme co-expression system, consisting of a catalase and an L-glutamic oxidase for the generation of 2OG, to convert penicillin G to G-7-ADCA in E. coli. The engineered whole-cell enzyme cascade was applied to an up-scaled reaction, exhibiting a yield of G-7-ADCA up to 39.21 mM (14.6 g ⋅ L-1 ) with a conversion of 78.42 mol %. This work highlights the potential of the integrated whole-cell system that may inspire further research on green and efficient production of 7-ADCA.

Biosynthesis of ß-lactam nuclei in yeast.

We have engineered brewer's yeast as a general platform for de novo synthesis of diverse ß-lactam nuclei starting from simple sugars, thereby enabling ready access to a number of structurally different antibiotics of significant pharmaceutical importance. The biosynthesis of ß-lactam nuclei has received much attention in recent years, while rational engineering of non-native antibiotics-producing microbes to produce ß-lactam nuclei remains challenging. Benefited by the integration of heterologous biosynthetic pathways and rationally designed enzymes that catalyze hydrolysis and ring expansion reactions, we succeeded in constructing synthetic yeast cell factories which produce antibiotic cephalosporin C (CPC, 170.1 ± 4.9 µg/g DCW) and the downstream ß-lactam nuclei, including 6-amino penicillanic acid (6-APA, 5.3 ± 0.2 mg/g DCW), 7-amino cephalosporanic acid (7-ACA, 6.2 ± 1.1 µg/g DCW) as well as 7-amino desacetoxy cephalosporanic acid (7-ADCA, 1.7 ± 0.1 mg/g DCW). This work established a Saccharomyces cerevisiae platform capable of synthesizing multiple ß-lactam nuclei by combining natural and artificial enzymes, which serves as a metabolic tool to produce valuable ß-lactam intermediates and new antibiotics.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Ferritin-Displayed GLP-1 with Improved Pharmacological Activities and Pharmacokinetics.

Glucagon-like peptide-1 (GLP-1) is an incretin (a type of metabolic hormone that stimulates a decrease in blood glucose levels), holding great potential for the treatment of type 2 diabetes mellitus (T2DM). However, its extremely short half-life of 1-2 min hampers any direct clinical application. Here, we describe the application of the heavy chain of human ferritin (HFt) nanocage as a carrier to improve the pharmacological properties of GLP-1. The GLP-HFt was designed by genetic fusion of GLP-1 to the N-terminus of HFt and was expressed in inclusion bodies in E. coli. The refolding process was developed to obtain a soluble GLP-HFt protein. The biophysical properties determined by size-exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD), transmission electron microscopy (TEM), and X-ray crystallography verified that the GLP-HFt successfully formed a 24-mer nanocage with GLP-1 displayed on the external surface of HFt. The in vivo pharmacodynamic results demonstrated that the GLP-HFt nanocage retained the bioactivity of natural GLP-1, significantly reduced the blood glucose levels for at least 24 h in a dose-dependent manner, and inhibited food intake for at least 8-10 h. The half-life of the GLP-HFt nanocage was approximately 52 h in mice after subcutaneous injection. The prolonged half-life and sustained control of blood glucose levels indicate that the GLP-HFt nanocage can be further developed for the treatment of T2DM. Meanwhile, the HFt nanocage proves its great potential as a universal carrier that improves the pharmacodynamic and pharmacokinetic properties of a wide range of therapeutic peptides and proteins.

Expression and purification of a difficult sarcomeric protein: Telethonin.

Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.

Thiamet G as a Potential Treatment for Polycystic Kidney Disease.

BACKGROUND/AIM: Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder primarily caused by mutations in Pkd1 (PC1), which account for the majority of ADPKD cases. These mutations contribute to the formation of cysts in the kidneys and other organs, ultimately leading to renal failure. Unfortunately, there are currently no available preventive treatments for this disease. MATERIALS AND METHODS: In this study, we utilized Pkd1-knockdown mice and cells to investigate the potential involvement of O-GlcNAcylation in the progression of PKD. Additionally, we examined the effects of thiamet G, an inhibitor of O-GlcNAcase (OGA), on PKD mice. RESULTS: Our findings indicate that both O-GlcNAcylation and OGT (O-GlcNAc transferase) were downregulated in the renal tissues of Pkd1-silenced mice. Furthermore, O-GlcNAcylation was shown to regulate the stability and function of the C-terminal cytoplasmic tail (CTT) of PC1. Treatment of PKD mice with thiamet G resulted in a reduction of renal cytogenesis in these animals. CONCLUSION: These results highlight the unique role of O-GlcNAcylation in the development of cyst formation in PKD and propose it as a potential therapeutic target for the treatment of PKD.

Generation of novel long-acting GLP-1R agonists using DARPins as a scaffold.

Glucagon-like peptide-1 (GLP-1) has been considered to be a promising peptide for treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life (minutes) of native GLP-1 limits its clinical application potential. Here, we designed two GLP-1 analogues by genetic fusion of GLP-1 to one or two tandem human serum albumin-binding designed ankyrin repeat proteins (DARPins), denoted as GLP-DARPin or GLP-2DARPin. The two DARPin-fusion GLP-1 proteins were expressed in E. coli and purified, followed by measurements of their bioactivities and half-lives in mice. The results revealed that the half-life of GLP-2DARPin, binding two HSA molecules, was approximately 3-fold longer than GLP-DARPin (52.3 h versus 18.0 h). In contrast, the bioactivity results demonstrated that the blood glucose-lowering effect of GLP-DARPin was more potent than that of GLP-2DARPin. The oral glucose tolerance tests indicated that blood glucose levels were significantly reduced for at least 48 h by GLP-DARPin, but were reduced for only 24 h by GLP-2DARPin. Injected once every two days, GLP-DARPin substantially reduced blood glucose levels in streptozotocin (STZ)-induced diabetic mice to the same levels as normal mice. During the treatment course, GLP-DARPin significantly reduced the food intake and body weight of diabetic mice up to approximately 17% compared with the control group. A histological analysis revealed that GLP-DARPin alleviated islet loss in diabetic mice. These findings suggest that long-acting GLP-DARPin holds great potential for further development into drugs for the treatment of T2DM and obesity. Meanwhile, our data indicate that albumin-binding DARPins can be used as a universal scaffold to improve the pharmacokinetic profiles and pharmacological activities of therapeutic peptides and proteins.

Albumin-binding domain extends half-life of glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) has been used widely for half-life extension of therapeutic peptides and proteins. In the present study, novel GLP-1 receptor agonists were designed by genetic fusion of GLP-1 to three kinds of ABDs with different affinities for HSA: GA3, ABD035 and ABDCon. The bioactivities and half-lives of ABD-fusion GLP-1 proteins with different types and lengths of linkers were investigated in vitro and in vivo. The results demonstrated that ABD-fusion GLP-1 proteins could bind to HSA with high affinity. The blood glucose-lowering effect of GLP-1 was significantly improved and sustained by fusion to ABD. Meanwhile, the fusion proteins significantly inhibited food intake, which was beneficial for T2DM and obesity treatment. The half-life of GLP-1 was substantially extended by virtue of ABD. The in vivo results also showed that a longer linker inserted between GLP-1 and ABD resulted in a higher blood glucose-lowering effect. The fusion proteins generated by fusion of GLP-1 to GA3, ABD035 and ABDCon exhibited similar bioactivities and pharmacokinetics in vivo. These findings demonstrate that ABD-fusion GLP-1 proteins retain the bioactivities of natural GLP-1 and can be further developed for T2DM treatment and weight loss. It also indicates that the ABD-fusion strategy can be generally applicable to any peptide or protein, to improve pharmacodynamic and pharmacokinetic properties.

Recent Advances in Half-life Extension Strategies for Therapeutic Peptides and Proteins.

Peptides and proteins are two classes of molecules with attractive possibilities for therapeutic applications. However, the bottleneck for the therapeutic application of many peptides and proteins is their short halflives in vivo, typically just a few minutes to hours. Half-life extension strategies have been extensively studied and many of them have been proven to be effective in the generation of long-acting therapeutics with improved pharmacokinetic and pharmacodynamic properties. In this review, we summarize the recent advances in half-life extension strategies, illustrate their potential applications and give some examples, highlighting the strategies that have been used in approved drugs and for drugs in clinical trials. Meanwhile, several novel strategies that are still in the process of discovery or at a preclinical stage are also introduced. In these strategies, the two most frequently used half-life extension methods are the reduction in the rate of renal clearance or the exploitation of the recycling mechanism of FcRn by binding to the albumin or IgG-Fc. Here, we discuss half-life extension strategies of recombinant therapeutic protein via genetic fusion, rather than chemical conjugation such as PEGylation. With the rapid development of genetic engineering and protein engineering, novel strategies for half-life extension have been emerged consistently. Some of these will be evaluated in clinical trials and may become viable alternatives to current strategies for making next-generation biodrugs.

Case-Control Study and Meta-Analysis Show a Weak Association between ANTXR2 Polymorphisms and Ankylosing Spondylitis in Chinese Han.

Previous studies have demonstrated associations of ANTXR2 gene polymorphisms with ankylosing spondylitis (AS). These associations differ depending on the ethnic populations and AS subgroups studied. Purposes of the current study were to evaluate the associations of 4 single nucleotide polymorphisms (SNPs) of the ANTXR2 gene with susceptibility to AS alone or AS in combination with acute anterior uveitis (AAU) in Chinese Han. Therefore, a case-control association study was performed in 880 AS+AAU-, 860 AS+AAU+, and 1700 healthy controls. Genotyping was performed using the iPLEXGold genotyping assay. Our results showed a weak association of rs6534639 AA genotype with AS+AAU+ patients (p=0.042), which was lost after correction for multiple comparisons. No other association was found between SNPs of ANTXR2 and susceptibility of AS+AAU- or AS+AAU+. A meta-analysis was performed to evaluate the associations of polymorphisms in the ANTXR2 gene with AS. Results showed a weak association of rs4389526 with AS susceptibility in all studies but failed to show an association of rs6534639 with AS in Chinese Han. Taken together, this study shows no association between ANTXR2 polymorphisms and AS susceptibility in a Chinese Han population, but meta-analysis showed that rs4389526 in the ANTXR2 gene was weakly associated with AS susceptibility in both Caucasian and Chinese Han patients.

Physiological signal analysis for fatigue level of experienced and inexperienced drivers.

OBJECTIVE: We studied the changes in driving fatigue levels of experienced and inexperienced drivers at 3 periods of the day: 9:00 a.m.-12:00 p.m., 12:00 p.m.-2:00 p.m., and 4:00 p.m.-6:00 p.m. METHODS: Thirty drivers were involved in 120-min real-car driving, and sleepiness ratings (Stanford Sleepiness Scale, SSS; Hoddes et al. 1973 ), electroencephalogram (EEG) signals, and heart rates (HRs) were recorded. Together with principal component analysis, the relationship between EEG signals and HR was explored and used to determine a comprehensive indicator of driving fatigue. Then the comprehensive indicator was assessed via paired t test. RESULTS: Experienced and inexperienced drivers behaved significantly differently in terms of subjective fatigue during preliminary trials. At the beginning of trials and after termination, subjective fatigue level was aggravated with prolonged continuous driving. Moreover, we discussed the changing rules of EEG signals and HR and found that with prolonged time, the ratios of δ and ß waves significantly declined, whereas that of the θ wave significantly rose. The ratio of (α + θ)/ß significantly rose both before trials and after termination, but HR dropped significantly. However, one-factor analysis of variance shows that driving experience significantly affects the θ wave, (α + θ)/ß ratio, and HR. CONCLUSIONS: We found that in a monotonous road environment, fatigue symptoms occurred in inexperienced drivers and experienced drivers after about 60 and 80 min of continuous driving, respectively. Therefore, as for drivers with different experiences, restriction on continuous driving time would avoid fatigued driving and thereby eliminate traffic accidents. We find that the comprehensive indicator changes significantly with fatigue level. The integration of different indicators improves the recognition accuracy of different driving fatigue levels.

Ocular Behcet's disease is associated with aberrant methylation of interferon regulatory factor 8 (IRF8) in monocyte-derived dendritic cells.

Aberrant methylation of interferon regulatory factor 8 (IRF8) has been noted in various tumors. IRF8 has also been reported to be involved in many autoimmune diseases, including Behcet's disease (BD). However, the methylation status of IRF8 in BD has not been reported. To address this issue, we investigated whether the degree of methylation of IRF8 in dendritic cells (DCs) plays a role in the development of BD. We found a lower mRNA expression and a higher methylation level of IRF8 in active ocular BD patients as compared to normal subjects and inactive patients. Treatment with a demethylation agent, 5-Aza-2'-deoxycytidine (DAC) resulted in an increase of mRNA expression and a reduction of the IRF8 methylation level. It also down-regulated the expression of the co-stimulatory molecules CD86, CD80, CD40, and reduced the production of IL-6, IL-1ß, IL-23 and IL-12. An inhibition of Th1/Th17 responses was observed as evidenced by a decreased production of IFN-γ, IL-17, and a reduction of IFN-γ/IL-17- producing CD4+ T cells following treatment with DAC. This study shows that active ocular BD patients have an aberrant IRF8 methylation status. These findings suggest that epigenetic control of IRF8 expression may offer a future target in the treatment of ocular BD.

Aberrant DNA methylation of GATA binding protein 3 (GATA3), interleukin-4 (IL-4), and transforming growth factor-ß (TGF-ß) promoters in Behcet's disease.

The pathogenesis of Behcet's disease (BD) remains poorly understood. The purpose of this study was to investigate whether an aberrant DNA methylation of transcriptional and inflammatory factors, including TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A and TGF-ß, in CD4+T confers risk to BD. We found that the promoter methylation level of GATA3, IL-4 and TGF-ß was significantly up-regulated in active BD patients and negatively correlated with the corresponding mRNA expression. The mRNA expression of GATA3 and TGF-ß was markedly down-regulated in active BD patients compared to healthy individuals. Treatment with corticosteroids and cyclosporine (CsA) resulted in a decrease of the methylation level of GATA3 and TGF-ß in inactive BD patients. Our results suggest that an aberrant DNA methylation of GATA3 and TGF-ß is associated with their mRNA expression and participates in the pathogenesis of BD.

Hypermethylation of Interferon Regulatory Factor 8 (IRF8) Confers Risk to Vogt-Koyanagi-Harada Disease.

Aberrant methylation change of IRF8 confers risk to various tumors, and abnormal expression of IRF8 is involved in many autoimmune diseases, including ocular Behcet's disease. However, whether the methylation change of IRF8 is associated with Vogt-Koyanagi-Harada (VKH) disease remains unknown. In the present study, we found a decreased IRF8 mRNA expression in association with a higher methylation level in monocyte-derived dendritic cells (DCs) from active VKH patients compared with the normal and inactive subjects. DCs incubated with cyclosporin a (CsA) or dexamethasone (DEX) showed a lower methylation and higher mRNA expression of IRF8 in active VKH patients. A demethylation reagent, 5-Aza-2'-deoxycytidine (DAC) showed a notable demethylation effect as evidenced by increasing the mRNA expression and reducing the methylation level of IRF8. It also suppressed the Th1 and Th17 responses through down-regulating the expression of co-stimulatory molecules (CD86, CD80, CD40), and reducing the production of pro-inflammatory cytokines (IL-6, IL-1ß, IL-23, IL-12) produced by DCs. These findings shows that hypermethylation of IRF8 in DCs confers risk to VKH disease. Demethylation of IRF8 may offer a novel therapeutic strategy protect against VKH disease.

Promoter Hypermethylation of GATA3, IL-4, and TGF-ß Confers Susceptibility to Vogt-Koyanagi-Harada Disease in Han Chinese.

Purpose: We investigated the role of promoter methylation of transcriptional and inflammatory factors, including TBX21, GATA3, RORγt, FOXP3, IFN-γ, IL-4, IL-17A, and TGF-ß in the development of Vogt-Koyanagi-Harada (VKH) disease. Methods: The promoter methylation levels were detected by the Sequenom MassARRAY system in CD4+ T cells that were separated from 20 healthy individuals and 32 VKH patients (20 in the active stage without medication, 12 in inactive stage with medication). The mRNA expression level of GATA3, IL-4, and TGF-ß in CD4+ T cells was analyzed by real-time RT-PCR. Results: The promoter methylation levels of GATA3, IL-4, and TGF-ß were significantly higher in active VKH patients than in healthy individuals (P < 0.05). A decreased mRNA expression of GATA3 and TGF-ß was found in active VKH patients, which was correlated negatively with the DNA methylation of these factors. Treatment with systemic corticosteroid and cyclosporin A (CsA) decreased the methylation level of GATA3 and TGF-ß in association with an increased mRNA expression of molecules and reduced disease activity. Conclusions: Our findings suggest that promoter hypermethylation of GATA3 and TGF-ß in CD4+ T cells confers risk to VKH disease in Han Chinese.

[Analysis of immobilized L-glutamate oxidase fused with cellulose binding domain on microcrystalline cellulose].

Immobilization of enzymes is important and widely applied in biocatalysis. Streptomyces platensis gene gox, encoding an extracellular L-glutamate oxidase (Gox), was fused to cellulose binding domain (CBDcex) from Cellulomonas fimi and the recombinant protein Gox-CBD was expressed in Escherichia coli. The fusion protein (Gox-CBD) was immobilized onto microcrystalline cellulose. The preparation conditions, binding capacity, properties and stability of the immobilized enzyme were studied. Under the condition of 4 ℃, for 1 hour, the fusion protein Gox-CBD was able to bind microcrystalline cellulose at a ratio of 9.0 mg of protein per gram of microcrystalline cellulose. Enzymatic properties of free and immobilized L-glutamic oxidase (Gox-CBD) were compared. The specific activity of the immobilized enzyme decreased, but its thermal stability increased a lot compared with that of the free Gox-CBD. After incubation at 60 ℃ for 30 min, 70% of the total activity remained whereas the free recombinant Gox completely lost its activity. The immobilized protein was tightly bound to microcrystalline cellulose at pH below 10 or more than 5 mmol/L NaCl. The fusion protein of Gox-CBD can be specifically immobilized on the microcrystalline cellulose on a single step. Therefore, our findings can provide a novel strategy for protein purification and enzyme immobilization.

Pleiotropic Effects of Myocardial MMP-9 Inhibition to Prevent Ventricular Arrhythmia.

Observational studies have established a strong association between matrix metalloproteinase-9 (MMP-9) and ventricular arrhythmia. However, whether MMP-9 has a causal link to ventricular arrhythmia, as well as the underlying mechanism, remains unclear. Here, we investigated the mechanistic involvement of myocardial MMP-9 in the pathophysiology of ventricular arrhythmia. Increased levels of myocardial MMP-9 are linked to ventricular arrhythmia attacks after angiotensin II (Ang II) treatment. MMP-9-deficient mice were protected from ventricular arrhythmia. Increased expressions of protein kinase A (PKA) and ryanodine receptor phosphorylation at serine 2808 (pS2808) were correlated with inducible ventricular arrhythmia. MMP-9 deficiency consistently prevented PKA and pS2808 increases after Ang II treatment and reduced ventricular arrhythmia. Calcium dynamics were examined via confocal imaging in isolated murine cardiomyocytes. MMP-9 inhibition prevents calcium leakage from the sarcoplasmic reticulum and reduces arrhythmia-like irregular calcium transients via protein kinase A and ryanodine receptor phosphorylation. Human induced pluripotent stem cell-derived cardiomyocytes similarly show that MMP-9 inhibition prevents abnormal calcium leakage. Myocardial MMP-9 inhibition prevents ventricular arrhythmia through pleiotropic effects, including the modulation of calcium homeostasis and reduced calcium leakage.

Ribavirin enhances the action of interferon-α against hepatitis C virus by promoting the p53 activity through the ERK1/2 pathway.

BACKGROUND/AIMS: Ribavirin significantly enhances the antiviral response of interferon-α (IFN-α) against Hepatitis C virus (HCV), but the underlying mechanisms remain poorly understood. Recently, p53 has been identified as an important factor involving the suppression of HCV replication in hepatocytes. We, therefore, decided to investigate whether and how ribavirin inhibits the replication of HCV by promoting the activity of p53. METHODS: HepG2 and HCV replicons (JFH1/HepG2) were utilized to study the relationship between ribavirin and p53. The effect of ribavirin on cell cycles was analyzed by flow cytometry. The activation of p53 and the signaling pathways were determined using immunoblotting. By knocking down ERK1/ERK2 and p53 utilizing RNA interference strategy, we further assessed the role of ERK1/2 and p53 in the suppression of HCV replication by ribavirin in a HCV replicon system. RESULTS: Using HepG2 and HCV replicons, we demonstrated that ribavirin caused the cell cycle arrest at G1 phase and stabilized and activated p53, which was associated with the antiviral activity of ribavirin. Compared to either ribavirin or IFN-α alone, ribavirin plus IFN-α resulted in greater p53 activation and HCV suppression. We further identified ERK1/2 that linked ribavirin signals to p53 activation. More importantly, knockdown of ERK1/2 and p53 partially mitigated the inhibitory effects of ribavirin on the HCV replication, indicating that ERK1/2-p53 pathway was involved in the anti-HCV effects of ribavirin. CONCLUSION: Ribavirin stimulates ERK1/2 and subsequently promotes p53 activity which at least partly contributes to the enhanced antiviral response of IFN-α plus ribavirin against HCV.

Ribavirin enhances interferon signaling via stimulation of mTOR and p53 activities.

Cellular mechanisms involving the enhancement of interferon (IFN) signaling by ribavirin remain poorly understood. Here, we identified a novel role of ribavirin in the communication between p53 and the mammalian target of rapamycin (mTOR) signaling. Ribavirin activates p53 by stimulating mTOR and promoting the interaction between mTOR and p53. Activated p53 stimulates the transcription of IFN regulatory factor 9 and subsequently enhances IFN signaling. Furthermore, ribavirin-induced activation of mTOR and p53 enhances IFN-dependent signaling for the IFN-alpha/ribavirin combined treatment. We conclude that ribavirin enhances activities of mTOR and p53, which may account for its antiviral and antitumor effects.

Ribavirin up-regulates the activity of double-stranded RNA-activated protein kinase and enhances the action of interferon-alpha against hepatitis C virus.

BACKGROUND: Ribavirin's mechanism of action in the treatment of chronic hepatitis C remains to be clarified. Double-stranded RNA-activated protein kinase (PKR) plays a role in cell defense against virus infection. This study investigated whether PKR is a mediator of the effectiveness of ribavirin, used either alone or in combination with interferon (IFN)- alpha , against hepatitis C virus (HCV) infection. METHODS: Primary human hepatocytes and HCV-replicon cells were treated with ribavirin and/or IFN- alpha . PKR activity was assayed by immunoblotting. A pulse-chase assay of the half-life of PKR protein was performed to study whether ribavirin decreases PKR degradation. We used small-interference RNA (siRNA) to knock down PKR to assess its importance in the suppression of HCV-RNA replication in the replicon system. RESULTS: Ribavirin was able to up-regulate the levels of phosphorylated PKR and phosphorylated eIF2 alpha , leading to suppression of HCV-RNA replication. The effects that treatment with ribavirin plus IFN- alpha had on PKR activity were greater than those observed for treatment with either ribavirin alone or IFN- alpha alone. Knockdown of PKR increased HCV-RNA replication, supporting the importance of PKR in the control of HCV-RNA replication. The pulse-chase experiment showed that ribavirin can reduce the degradation rate of PKR protein. CONCLUSION: These results suggest that the anti-HCV action of ribavirin is partly attributable to its ability to up-regulate PKR activity.