Functionalization of rice husk-derived mesoporous silica nanoparticles for targeted and imaging in cancer drug delivery.

BACKGROUND: Rice, a pivotal global food staple, annually accumulates vast amounts of rice husks, resulting in substantial environmental impact. Exploiting the high silica content in rice husk, our research aimed to recycle this agricultural byproduct to synthesize mesoporous silica nanoparticles (rMSNs). These nanoparticles were further modified to evaluate their potential as effective carriers for cancer drug delivery. RESULTS: rMSNs showed high biocompatibility, large surface area and porous structure as MSNs, making them excellent drug carriers. Further modifications were applied to rMSNs, such as the incorporation of the lanthanides europium and gadolinium into rMSNs, making them fluorescent and magnetic for detection and tracking using confocal fluorescence microscopy and magnetic resonance imaging. Additionally, folic acid and aptamer AS1411 were conjugated with rMSNs to enhance the targeting of cancer cells. HeLa cells exhibited higher uptake of camptothecin (CPT)-loaded rMSNs compared to normal fibroblast cells (L929). The linkage of disulfide bonds to rMSNs also allowed CPT to be carried by rMSNs and released intracellularly in the presence of the abundant reducing agent glutathione. The validation of rMSNs in vitro and in vivo proved their practical feasibility. CONCLUSION: Our findings indicate that low-cost rMSNs, derived from recycled agricultural waste, can replace highly valuable MSNs. Functionalized rMSNs exhibit promising capabilities in transporting clinical drugs to specific aberrant tissues and offering dual-targeting and dual-imaging functionalities for enhanced cancer therapy. © 2023 Society of Chemical Industry.

Distinctive microbial community and genome structure in coastal seawater from a human-made port and nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean.

Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.

Comparative Transcriptome Analysis of Organ-Specific Adaptive Responses to Hypoxia Provides Insights to Human Diseases.

The common carp is a hypoxia-tolerant fish, and the understanding of its ability to live in low-oxygen environments has been applied to human health issues such as cancer and neuron degeneration. Here, we investigated differential gene expression changes during hypoxia in five common carp organs including the brain, the gill, the head kidney, the liver, and the intestine. Based on RNA sequencing, gene expression changes under hypoxic conditions were detected in over 1800 genes in common carp. The analysis of these genes further revealed that all five organs had high expression-specific properties. According to the results of the GO and KEGG, the pathways involved in the adaptation to hypoxia provided information on responses specific to each organ in low oxygen, such as glucose metabolism and energy usage, cholesterol synthesis, cell cycle, circadian rhythm, and dopamine activation. DisGeNET analysis showed that some human diseases such as cancer, diabetes, epilepsy, metabolism diseases, and social ability disorders were related to hypoxia-regulated genes. Our results suggested that common carp undergo various gene regulations in different organs under hypoxic conditions, and integrative bioinformatics may provide some potential targets for advancing disease research.

Microencapsulated Recombinant Human Epidermal Growth Factor Ameliorates Osteoarthritis in a Murine Model.

Osteoarthritis, a highly age-related and chronic inflammatory disorder with cartilage loss, causes patients difficultly in movement; there is no efficient and sustainable remedy for osteoarthritis currently. Although hyaluronic acid (HA) and platelet-rich plasma (PRP) have been used to alleviate osteoarthritis, the effects could be short and multiple injections might be required. To address this issue, we exploited the property of chitosan to encapsulate recombinant human epidermal growth factor and obtained microencapsulated rhEGF (Me-rhEGF). In the current study, we induced the osteoarthritis-like symptoms with monosodium iodoacetate (MIA) in rats and investigated the therapeutic effects of Me-rhEGF. Following administration of HA/Me-rhEGF in vivo, we observed that the total Mankin scores, cartilage oligomeric protein, C-telopeptide of type II collagen, IL-1ß, IL-6, IL-17A, and TNF-α cytokines, nitric oxide, and prostaglandin E2 expressions were significantly inhibited. Our results also strongly indicate that individual use of HA or rhEGF slightly decreased the inflammation and restored the destructive joint structure, but was not as drastic as seen in the HA/Me-rhEGF. Moreover, HA/Me-rhEGF profoundly reduced cartilage destruction and proteoglycan loss and downregulated matrix metalloproteinase expressions. These findings reveal that the treatment of HA/Me-rhEGF could be more beneficial than the use of single HA or rhEGF in reliving osteoarthritis and demonstrate the therapeutic application of microencapsulation technology in difficult joint disorders. In essence, we believe that the Me-rhEGF could be promising for further research and development as a clinical treatment against osteoarthritis.

Hesperidin and Chlorogenic Acid Synergistically Inhibit the Growth of Breast Cancer Cells via Estrogen Receptor/Mitochondrial Pathway.

Breast cancer is the most common cancer in women worldwide. Hesperidin (Hes) and chlorogenic acid (CA) are traditional medicinal molecules that abundantly exist in natural plants or foods. These compounds have been shown to prevent and suppress various cancers and therefore can be utilized as adjunctive therapies to aid cancer treatment. Here, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays show a greater synergistic inhibitory effect on the growth of breast cancer cells, MCF-7, but not normal breast cells, MCF-10A, than hesperidin or chlorogenic acid alone. We present the possible molecular signaling pathways in MCF-7 cells with or without herbal molecule treatments via proteomic approaches. The data were further analyzed by Ingenuity Pathway Analysis (IPA) and confirmed by quantifying mRNA associated with the estrogen-receptor signaling pathway and mitochondrial functions. We demonstrated that the expression of CYC1, TFAM, ATP5PB, mtATP6, mtDNA, and NRF-1 were decreased upon 12 h treatment, and subsequent ATP production was also significantly decreased at 24 h. These results identified a synergistic effect induced by combinational treatment with hesperidin and chlorogenic acid, which can regulate mitochondria and ATP production through the estrogen receptor pathway in MCF-7 cells. However, none of the treatments induced the generation of reactive oxygen species (ROS), suggesting that ROS likely plays no role in the observed pharmacological activities. Overall, our study sheds light on the adequacy of hesperidin and chlorogenic acid to serve as an adjunctive therapy when co-administrated with chemotherapy drugs in breast cancer patients.

A Natural Botanical Product, Resveratrol, Effectively Suppresses Vesicular Stomatitis Virus Infection In Vitro.

Numerous natural phytochemicals such as resveratrol are acknowledged as potent botanical agents in regulating immune responses. However, it is less understood whether such immunomodulatory phytochemicals are appropriate for use as direct treatments in veterinary viral diseases. In the present study, we investigated the efficacy of resveratrol in suppressing vesicular stomatitis virus (VSV) infection. Outbreaks of VSV can cause massive economic loss in poultry and livestock husbandry farming, and VSV treatment is in need of therapeutic development. We utilized a recombinant VSV that expresses green fluorescent protein (GFP) to measure viral replication in cells treated with resveratrol. Our findings revealed that resveratrol treatment affords a protective effect, shown by increased viability and reduced viral replication, as indicated by a reduction in fluorescent signals. Additionally, we found that resveratrol inhibition of VSV infection occurs via suppression of the caspase cascade. Structural analysis also indicated that resveratrol potentially interacts with the active sites of caspase-3 and -7, facilitating antiviral activity. The potential effect of resveratrol on reducing VSV infection in vitro suggests that resveratrol should be further investigated as a potential veterinary therapeutic or prophylactic agent.

High Amplification of the Antiviral Activity of Curcumin through Transformation into Carbon Quantum Dots.

It is demonstrated that carbon quantum dots derived from curcumin (Cur-CQDs) through one-step dry heating are effective antiviral agents against enterovirus 71 (EV71). The surface properties of Cur-CQDs, as well as their antiviral activity, are highly dependent on the heating temperature during synthesis. The one-step heating of curcumin at 180 °C preserves many of the moieties of polymeric curcumin on the surfaces of the as-synthesized Cur-CQDs, resulting in superior antiviral characteristics. It is proposed that curcumin undergoes a series of structural changes through dehydration, polymerization, and carbonization to form core-shell CQDs whose surfaces remain a pyrolytic curcumin-like polymer, boosting the antiviral activity. The results reveal that curcumin possesses insignificant inhibitory activity against EV71 infection in RD cells [half-maximal effective concentration (EC50 ) >200 µg mL-1 ] but exhibits high cytotoxicity toward RD cells (half-maximal cytotoxic concentration (CC50 ) <13 µg mL-1 ). The EC50 (0.2 µg mL-1 ) and CC50 (452.2 µg mL-1 ) of Cur-CQDs are >1000-fold lower and >34-fold higher, respectively, than those of curcumin, demonstrating their far superior antiviral capabilities and high biocompatibility. In vivo, intraperitoneal administration of Cur-CQDs significantly decreases mortality and provides protection against virus-induced hind-limb paralysis in new-born mice challenged with a lethal dose of EV71.

Ultrastrong trapping of VEGF by graphene oxide: Anti-angiogenesis application.

Angiogenesis is the process of formation of new blood vessels, which is essential to human biology, and also plays a crucial role in several pathologies such as tumor growth and metastasis, exudative age-related macular degeneration, and ischemia. Vascular endothelial growth factor (VEGF), in particular, VEGF-A165 is the most important pro-angiogenic factor for angiogenesis. Thus, blocking the interaction between VEGFs and their receptors is considered an effective anti-angiogenic strategy. We demonstrate for that first time that bovine serum albumin-capped graphene oxide (BSA-GO) exhibits high stability in physiological saline solution and possesses ultrastrong binding affinity towards VEGF-A165 [dissociation constant (Kd) ∼3 × 10-12 M], which is at least five orders of magnitude stronger than that of high-abundant plasma proteins such as human serum albumin, fibrinogen, transferrin, and immunoglobulin G. Due to the surprising binding specificity of BSA-GO for VEGF-A165 in complex plasma fluid, we have also studied the anti-angiogenic effects in vitro and in vivo. Results show that BSA-GO not only effectively inhibits the proliferation, migration and tube formation of human umbilical vein endothelial cells, but also strongly disturbs the physiological process of angiogenesis in chick chorioallantoic membrane and blocks VEGF-A165-induced blood vessel formation in rabbit corneal neovascularization. Our findings indicate that GO nanomaterials can potentially act as therapeutic anti-angiogenic agents via ultrastrong VEGF adsorption and its activity suppression.

Clozapine induces autophagic cell death in non-small cell lung cancer cells.

BACKGROUND/AIMS: Previous studies have shown that patients with schizophrenia have a lower incidence of cancer than the general population, and several antipsychotics have been demonstrated to have cytotoxic effects on cancer cells. However, the mechanisms underlying these results remain unclear. The present study aimed to investigate the effect of clozapine, which is often used to treat patients with refractory schizophrenia, on the growth of non-small cell lung carcinoma cell lines and to examine whether autophagy contributes to its effects. METHODS: A549 and H1299 cells were treated with clozapine, and cell cytotoxicity, cell cycle and autophagy were then assessed. The autophagy inhibitor bafilomycin A1 and siRNA-targeted Atg7 were used to determine the role of autophagy in the effect of clozapine. RESULTS: Clozapine inhibited A549 and H1299 proliferation and increased p21 and p27 expression levels, leading to cell cycle arrest. Clozapine also induced a high level of autophagy, but not apoptosis, in both cell lines, and the growth inhibitory effect of clozapine was blunted by treatment with the autophagy inhibitor bafilomycin A1 or with an siRNA targeting atg7. CONCLUSIONS: Clozapine inhibits cell proliferation by inducing autophagic cell death in two non-small cell lung carcinoma cell lines. These findings may provide insights into the relationship between clozapine use and the lower incidence of lung cancer among patients with schizophrenia.

Taurine prevented cell cycle arrest and restored neurotrophic gene expression in arsenite-treated SH-SY5Y cells.

The study investigated the effect of taurine on cell viability and neurotrophic gene expression in arsenite-treated human neuroblastoma SH-SY5Y cells. Arsenite-induced intracellular reactive oxygen species (ROS) and interrupted cell cycle in SH-SY5Y cells. In addition, arsenite reduced mitochondria membrane potential (MMP) and decreased neurotrophic gene expressions such as n-myc downstream-regulated gene 4 (NDRG-4), brain-derived neurotrophic factor (BDNF) and sirtuin-1 (SIRT-1) in SH-SY5Y cells. In parallel, taurine prevented cell cycle, restored MMP and reduced the intracellular ROS level, and taurine recovered NDRG-4, BDNF and SIRT-1 gene expressions in arsenite-treated SH-SY5Y cells while taurine alone has no effect on these parameters.

Pre-existing Fas ligand (FasL) in cancer cells elicits tumor-specific protective immunity, but delayed induction of FasL expression after inoculation facilitates tumor formation.

Overexpression of Fas ligand (FasL) in cancer cells elicits potential antitumor effects via recruitment of neutrophils. Conversely, FasL-expressing tumors may counterattack tumor-infiltrating lymphocytes by delivering apoptotic death signals via Fas/FasL interactions, which may lead to tumor escape. In order to distinguish the role of FasL in antitumor activity and tumor progression, Lewis lung carcinoma cells (LLC-1) were used to establish the cell line LLC-FasL, in which FasL expression was repressed by doxycycline (Dox) treatment and induced in the absence of Dox. LLC-FasL cells promote tumor regression when expressing FasL, whereas tumor outgrowth is observed by depletion of FasL expression. To investigate whether initial expression of FasL during tumor formation is critical for FasL-mediated tumor regression, Dox-treated LLC-FasL cells were inoculated into Dox-treated mice, but Dox treatment was stopped 5 days after inoculation. When low cell numbers were inoculated, we observed 80% survival and no tumor formation, whereas no mice survived inoculation with high cell numbers, despite the delayed induction of FasL by Dox withdrawal. The inoculation of a high density of cells may establish a favorable tumor microenvironment before the expression of FasL. Our findings demonstrate that FasL may elicit antitumor activity when it is initially present on injected cancer cells and thus can act prior to tumor microenvironment formation. Furthermore, a well-established tumor microenvironment abrogates FasL-mediated antitumor activity.

Convergent evidence from mouse and human studies suggests the involvement of zinc finger protein 326 gene in antidepressant treatment response.

OBJECTIVES: The forced swim test (FST) is a commonly used model to predict antidepressant efficacy. Uncovering the genetic basis of the model may unravel the mechanism of antidepressant treatment. METHODS: FVB/NJ (FVB) and C57BL/6J (B6) were first identified as the response and non-response strains to fluoxetine (a serotonin-specific reuptake inhibitor antidepressant) treatment in the mouse FST. Simple-interval (SIM) and composite-interval (CIM) mappings were applied to map the quantitative trait loci (QTLs) of the anti-immobility effect of fluoxetine in FST (FST(FLX)) in 865 male B6×FVB-F2 mice. The brain mRNA expressions of the gene with the maximum QTL-linkage signal for FST(FLX) after the FST were compared between B6 and FVB mice and also compared between fluoxetine and saline treatment. The association of the variants in the human homologue of the mouse FST(FLX)-QTL gene with major depressive disorder (MDD) and antidepressant response were investigated in 1080 human subjects (MDD/control = 582/498). RESULTS: One linkage signal for FST(FLX)-QTL was detected at an intronic SNP (rs6215396) of the mouse Zfp326 gene (maximal CIM-LOD = 9.36). The Zfp326 mRNA expression in the FVB thalamus was significantly down-regulated by fluoxetine in the FST, and the higher FVB-to-B6 Zfp326 mRNA expressions in the frontal cortex, striatum and hypothalamus diminished after fluoxetine treatment. Two coding-synonymous SNPs (rs2816881 and rs10922744) in the human homologue of Zfp326, ZNF326, were significantly associated with the 8-week antidepressant treatment response in the MDD patients (Bonferroni-corrected p = 0.004-0.028). CONCLUSIONS: The findings suggest the involvement of the Zfp326 and ZNF326 genes in antidepressant treatment response.

Autocrine CCL2 promotes cell migration and invasion via PKC activation and tyrosine phosphorylation of paxillin in bladder cancer cells.

The amount of monocyte chemoattractant protein-1 (MCP-1/CCL2) produced by a transitional cell carcinoma is directly correlated with high recurrence and poor prognosis in bladder cancer. However, the mechanisms underlying the effects of CCL2 on tumor progression remain unexplored. To investigate the role played by CCL2, we examined cell migration in various bladder cancer cell lines. We found that high-grade cancer cells expressing high levels of CCL2 showed more migration activity than low-grade bladder cancer cells expressing low levels of the chemokine. Although the activation of CCL2/CCR2 signals did not appreciably affect cell growth, it mediated cell migration and invasion via the activation of protein kinase C and phosphorylation of tyrosine in paxillin. Blocking CCL2 and CCR2 with small hairpin RNA (shCCL2) or a specific inhibitor reduced CCL2/CCR2-mediated cell migration. The antagonist of CCR2 promoted the survival of mice bearing MBT2 bladder cancer cells, and CCL2-depleted cells showed low tumorigenicity compared with shGFP cells. In addition to observing high-levels of CCL2 in high-grade human bladder cancer cells, we showed that the CCL2/CCR2 signaling pathway mediated migratory and invasive activity, whereas blocking the pathway decreased migration and invasion. In conclusion, high levels of CCL2 expressed in bladder cancer mediates tumor invasion and is involved with advanced tumorigenesis. Our findings suggest that this CCL2/CCR2 pathway is a potential candidate for the attenuation of bladder cancer metastases.

C-terminal region of Candida rugosa lipases affects enzyme activity and interfacial activation.

Candida rugosa contains several lipase (CRLs) genes, and CRLs show diverse enzyme activity despite being highly homologous across their entire protein family. Previous studies found that LIP4 has a high esterase activity and a low lipolytic activity and lacks interfacial activation. To investigate whether the C-terminal region of the CRLs mediates enzymatic activity, chimeras were generated in which the C-terminus of LIP4 from either residue 374, 396, 417, or 444 to residue 534 was swapped with the corresponding peptide from the isoform LIP1. A chimeric lipase containing the C-terminus from 396 to 534 of LIP1 on a LIP4 scaffold showed activity similar to that of commercial CRL on triolein, and lipolytic activity increased 2-6-fold over that of LIP4. Moreover, interfacial activation was also observed in the chimeric lipase. To improve its enzymatic properties, a novel glycosylation site was added at residue 314. The new glycosylated lipase showed improved thermostability and enhancement in enzymatic activity, indicating its potential for use in further application.

Evidence of involvement of the human Par-4 (PAWR) gene in major depressive disorder.

UNLABELLED: QBJECTIVES: The aim of the study was to examine the associations between genetic variations in the human PAWR gene and major depressive disorder (MDD) as well as the response to antidepressant treatment. METHODS: Six-hundred and two patients with MDD and 543 controls were included in the study; among the MDD patients, 268 were followed-up for a further 8 weeks in order to assess their response to treatment with selective serotonin reuptake inhibitors (SSRIs). Six polymorphisms (rs17005769, rs4842318, rs7305141, rs2307223, rs8176874 and rs2307220) of the PAWR gene were investigated with regard to their association with MDD and antidepressant treatment efficacy. RESULTS: One polymorphism, rs8176874, was in genotypic (uncorrected P=0.005) and allelic (uncorrected P=0.0015) association with MDD. Several haplotypes spanning rs7305141-rs2307223-rs8176874 were also significantly associated with MDD after correction for multiple testing (corrected P<0.05). However, neither single-marker nor haplotype-based analyses suggested an association between the studied markers and SSRI treatment response. CONCLUSIONS: Genetic variations in the PAWR gene are related to susceptibility to MDD but not to SSRI treatment response.

ENU mutagenesis identifies mice with morbid obesity and severe hyperinsulinemia caused by a novel mutation in leptin.

BACKGROUND: Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. Leptin is central to the regulation of energy metabolism and control of body weight in mammals. METHODOLOGY/PRINCIPAL FINDINGS: To better recapitulate the complexity of human obesity syndrome, we applied N-ethyl-N-nitrosourea (ENU) mutagenesis in combination with a set of metabolic assays in screening mice for obesity. Mapping revealed linkage to the chromosome 6 within a region containing mouse Leptin gene. Sequencing on the candidate genes identified a novel T-to-A mutation in the third exon of Leptin gene, which translates to a V145E amino acid exchange in the leptin propeptide. Homozygous Leptin(145E/145E) mutant mice exhibited morbid obesity, accompanied by adipose hypertrophy, energy imbalance, and liver steatosis. This was further associated with severe insulin resistance, hyperinsulinemia, dyslipidemia, and hyperleptinemia, characteristics of human obesity syndrome. Hypothalamic leptin actions in inhibition of orexigenic peptides NPY and AgRP and induction of SOCS1 and SOCS3 were attenuated in Leptin(145E/145E) mice. Administration of exogenous wild-type leptin attenuated hyperphagia and body weight increase in Leptin(145E/145E) mice. However, mutant V145E leptin coimmunoprecipitated with leptin receptor, suggesting that the V145E mutation does not affect the binding of leptin to its receptor. Molecular modeling predicted that the mutated residue would form hydrogen bond with the adjacent residues, potentially affecting the structure and formation of an active complex with leptin receptor within that region. CONCLUSIONS/SIGNIFICANCE: Thus, our evolutionary, structural, and in vivo metabolic information suggests the residue 145 as of special function significance. The mouse model harboring leptin V145E mutation will provide new information on the current understanding of leptin biology and novel mouse model for the study of human obesity syndrome.

Improved body weight and metabolic outcomes in overweight or obese psychiatric patients switched to amisulpride from other atypical antipsychotics.

Switching to a different second-generation antipsychotic (SGA) with a lower risk of weight gain is recommended for overweight or obese psychiatric patients undergoing SGA treatment. However, there have been no complete reports regarding the long-term metabolic effects of switching to amisulpride. In this open-label 1-year study, we investigated the effects on body weight and other metabolic profiles when psychiatric patients treated with another SGA were switched to amisulpride treatment. Forty-six schizophrenia or schizoaffective inpatients with a body mass index greater than 27 kg/m were enrolled in the switch group. These patients were cross-titrated to amisulpride treatment and followed up for 1 year prospectively. Another 46 inpatients matched with the baseline body mass index of those in the switch group were enrolled as the control group retrospectively. The results showed that the switch group had greater weight loss than the control group (7.80 +/- 6.67 vs 2.60 +/- 6.23 kg, respectively; repeated-measure analysis of variance, P < 0.0005). During the treatment course, the amisulpride-treated patients showed significantly decreased fasting triglyceride, total cholesterol, glucose, and insulin resistance levels; decreased diastolic blood pressure and pulse rate; and a significant increase in high-density lipoprotein cholesterol levels after switching to amisulpride (all with a P < 0.05). The prevalence of metabolic syndrome in amisulpride-treated patients also decreased significantly from 65.2% to 30.4% (McNemar test, P < 0.0005). These findings suggest that switching to amisulpride could be an effective treatment of overweight or obese psychiatric patients treated previously with other SGAs.

Modulation of p53 transcription regulatory activity and post-translational modification by hepatitis C virus core protein.

Oncogenic virus proteins often target to tumor suppressor p53 during virus life cycle. In the case of hepatitis C virus (HCV) core protein, it has been shown to affect p53-dependent transcription. Here, we further characterized the in vitro and in vivo interactions between HCV core protein and p53 and showed that these two proteins colocalized in subnuclear granular structures and the perinuclear area. By use of a reporter assay, we observed that while low level of HCV core protein enhanced the transactivational activity of p53, high level of HCV core protein inhibited this activity. In both cases, however, HCV core protein increased the p53 DNA-binding affinity in gel retardation analyses, likely due to the hyperacetylation of p53 Lys(373) and Lys(382) residues. Additionally, HCV core protein, depending on its expression level, had differential effects on the Ser(15) phosphorylation of p53. Moreover, HCV core protein could rescue p53-mediated suppressive effects on both RNA polymerase I and III transcriptions. Collectively, our results indicate that HCV core protein targets to p53 pathway via at least three means: physical interaction, modulation of p53 gene regulatory activity and post-translational modification. This feature of HCV core protein, may potentially contribute to the HCV-associated pathogenesis.

Activation of RNA polymerase I transcription by hepatitis C virus core protein.

The hepatitis C virus (HCV) core protein has been implicated in the transregulation of various RNA polymerase (Pol) II dependent genes as well as in the control of cellular growth and proliferation. In this study, we show that the core protein, whether individually expressed or produced as part of the HCV viral polyprotein, is the only viral product that has the potential to activate RNA Pol I transcription. Deletion analysis demonstrated that the fragment containing the N-terminal 1-156 residues, but not the 1-122 residues, of HCV core protein confers the same level of transactivation activity as the full-length protein. Moreover, the integrity of the Ser(116) and Arg(117) residues of HCV core protein was found to be critical for its transregulatory functions. We used DNA affinity chromatography to analyze the human ribosomal RNA promoter associated transcription machinery, and the results indicated that recruitment of the upstream binding factor and RNA Pol I to the ribosomal RNA promoter is enhanced in the presence of HCV core protein. Additionally, the HCV core protein mediated activation of ribosomal RNA transcription is accompanied by the hyperphosphorylation of upstream binding factor on serine residues, but not on threonine residues. Moreover, HCV core protein is present within the RNA Pol I multiprotein complex, indicating its direct involvement in facilitating the formation of a functional transcription complex. Protein-protein interaction studies further indicated that HCV core protein can associate with the selectivity factor (SL1) via direct contact with a specific component, TATA-binding protein (TBP). Additionally, the HCV core protein in cooperation with TBP is able to activate RNA Pol II and Pol III mediated transcription, in addition to RNA Pol I transcription. Thus, the results of this study suggest that HCV has evolved a mechanism to deregulate all three nuclear transcription systems, partly through targeting of the common transcription factor, TBP. Notably, the ability of the HCV core protein to upregulate RNA Pol I and Pol III transcription supports its active role in promoting cell growth, proliferation, and the progression of liver carcinogenesis during HCV infection.

Mechanisms for inhibition of hepatitis B virus gene expression and replication by hepatitis C virus core protein.

We have demonstrated previously that the core protein of hepatitis C virus (HCV) exhibits suppression activity on gene expression and replication of hepatitis B virus (HBV). Here we further elucidated the suppression mechanism of HCV core protein. We demonstrated that HCV core protein retained the inhibitory effect on HBV gene expression and replication when expressed as part of the full length of HCV polyprotein. Based on the substitution mutational analysis, our results suggested that mutation introduced into the bipartite nuclear localization signal of the HCV core protein resulted in the cytoplasmic localization of core protein but did not affect its suppression ability on HBV gene expression. Mutational studies also indicated that almost all dibasic residue mutations within the N-terminal 101-amino acid segment of the HCV core protein (except Arg(39)-Arg(40)) impaired the suppression activity on HBV replication but not HBV gene expression. The integrity of Arg residues at positions 101, 113, 114, and 115 was found to be essential for both suppressive effects, whereas the Arg residue at position 104 was important only in the suppression of HBV gene expression. Moreover, our results indicated that the suppression on HBV gene expression was mediated through the direct interaction of HCV core protein with the trans-activator HBx protein, whereas the suppression of HBV replication involved the complex formation between HBV polymerase (pol) and the HCV core protein, resulting in the structural incompetence for the HBV pol to bind the package signal and consequently abolished the formation of the HBV virion. Altogether, this study suggests that these two suppression effects on HBV elicited by the HCV core protein likely depend on different structural context but not on nuclear localization of the core protein, and the two effects can be decoupled as revealed by its differential targets (HBx or HBV pol) on these two processes of the HBV life cycle.