Hyune Mo Rho

Transcriptional Activation of the Cu,Zn-Superoxide Dismutase Gene through the AP2 Site by Ginsenoside Rb2 Extracted from a Medicinal Plant, Panax ginseng

We report here that the ginseng saponins induce the transcription of Cu,Zn-superoxide dismutase gene (SOD1), which is one of the major antioxidant enzymes. Total saponins and panaxatriol did not elevate the level of SOD1, but panaxadiol significantly increased SOD1. Among the panaxadiol fractions, ginsenoside Rb2 was a more specific and more remarkable inducer of the SOD1 gene than ginsenoside Rb1. Deletion analyses of the SOD1 promoter revealed that the proximal promoter is responsible for this induction. Mobility shift assays with cis-elements in the proximal promoter region showed that specific binding of the AP2 transcription factor was significantly increased by treatment with ginsenoside Rb2. Mutations of the AP2 binding sites in the heterologous promoter and natural context systems abolished the transcriptional activation by ginsenoside Rb2. These results suggest that the SOD1 gene was greatly activated by ginsenoside Rb2 through transcription factor AP2 binding sites and its induction.

Transcriptional repression of the human p53 gene by hepatitis B viral X protein.

Hepatitis B viral X protein (HBx) and the human p53 protein (p53) have been known as a transactivator and as a tumor suppressor, respectively. These two proteins have also been known to interact with each other to neutralize their authentic functions and the p53 represses the HBV enhancer/X promoter activity. Here we report that the promoter activity of the human p53 gene was strongly repressed by the HBx using the chloramphenicol acetyl transferase (CAT) assay. Analyses of serial deletion, site-directed mutagenesis and the heterologous promoter system showed that the site responsible for the repression was the E-box element in the promoter of the p53 gene. In addition, HBx as expected also repressed the activation of the p53 promoter by c-Myc through the E-box element. Northern blot analyses also showed that the expression of the p53 gene in the HepG2-K8 cell line, which expresses HBV genes including HBx, was much more repressed than that of the control cell HepG2. These results with previous data suggest that the shift of the reciprocal inhibitory activities at the levels of protein-protein interaction and transcription between HBx and p53 may play a decisive role in the HBV-related hepatocarcinogenesis.

The activation of the rat copper/zinc superoxide dismutase gene by hydrogen peroxide through the hydrogen peroxide-responsive element and by paraquat and heat shock through the same heat shock element.

Copper/zinc superoxide dismutase (SOD1) protects cells against oxidative hazards by the dismutation of superoxide radicals. The promoter activity of theSOD1 gene was increased 3–5-fold by hydrogen peroxide, paraquat (PQ) and heat shock. Functional analyses of the regulatory region of the SOD1 gene by deletions, mutations, and heterologous promoter systems confirmed the induction of theSOD1 gene by H2O2 through the hydrogen peroxide-responsive element (HRE) (between nucleotides −533 and −520). Gel mobility shift assays showed that the existence of an H2O2-inducible protein bound to the oligonucleotide of the HRE. Similar analyses showed that the heat shock activated the SOD1 promoter through the heat shock element (HSE) (between nucleotides −185 and −171). A strong specific far-shifted complex with the oligonucleotide of the HSE was observed by the treatment of heat shock. When cells were treated with PQ, a strong far-shifted complex with the HSE was observed and was competed out by the cold HSE probe, indicating that PQ also activated theSOD1 promoter through the same HSE site. It is very interesting to note that chemical and physical stresses, such as PQ and heat shock, respectively, activated the SOD1 promoter through the same cis-element HSE. These results indicate that the SOD1 was inducible by H2O2through the HRE and by PQ and heat shock through the same HSE to protect cells from oxidative hazards.

The transcriptional activation of the human copper/zinc superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through two different regulator sites, the antioxidant responsive element and xenobiotic responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces SOD1 in human liver cells. Deletion analyses showed that the promoter region between −400 and −239 was responsible for the induction, in which two different characteristic regulatory elements, the antioxidant responsive element (ARE) and xenobiotic responsive element (XRE), are located. When the cells transfected with the plasmid containing those two cis-elements, the transactivation of SOD1 promoter was about 4-fold by TCDD, whereas mutation either on the ARE or XRE elevated the promoter activity by about 2-fold. Functional analyses of these two elements by deletion, mutation in the natural context, heterologous promoter assay, and gel mobility shift assay supported the notion that the activation of the SOD1 promoter was induced by TCDD through these two regulatory elements ARE and XRE. These results alongside our previous data indicate that the induction of SOD1 in response to TCDD is mediated by either Nrf2 protein or Ah receptor protein through ARE and XRE, respectively. These results also imply that the SOD1 can be induced by dioxin either in combination with or independently of these two regulatory elements to effectively defend cells from oxidative stress.

Interaction of hepatitis B viral X protein and CCAAT/enhancer-binding protein alpha synergistically activates the hepatitis B viral enhancer II/pregenomic promoter

The hepatitis B viral X protein (HBx) is known to exert its transactivation activity by the interaction with several cellular transcription factors. Here we report the interaction of HBx and CCAAT/enhancer-binding protein α (C/EBPα) and their effects on the enhancer/promoters of hepatitis B virus (HBV). A chloramphenicol acetyltransferase assay showed that the cotransfection of HBx and C/EBPα strongly activated the enhancer II/pregenomic promoter of HBV in a synergistic manner. This effect was also observed in the heterologous expression system with promoters of SV40 and herpes simplex virus thymidine kinase genes. Serial deletion analysis of the enhancer II/pregenomic promoter identified the responsible region (nucleotides 1639–1679), in which two C/EBP-binding sites are located. An in vitro interaction assay and electrophoretic mobility shift assay showed that HBx augmented the DNA binding activity of C/EBPα by direct interaction with it, and its basic leucine zipper domain was responsible for the interaction with HBx. Domain analysis of HBx showed that the central region (amino acids 78–103) was necessary for direct interaction with C/EBPα. However, the complete form of HBx was necessary for the synergistic activation of the HBV pregenomic promoter. These results suggest that the interaction of HBx and C/EBPα enhances the transcription of the HBV pregenomic promoter for the effective life cycle of HBV in hepatocytes.

Transcriptional activation of Cu/Zn superoxide dismutase and catalase genes by panaxadiol ginsenosides extracted from Panax ginseng.

Superoxide dismutase (SOD) converts superoxide radical to H2O2, which is in turn broken down to water and oxygen by catalase. Thus, SOD and catalase constitute the first coordinated unit of defence against reactive oxygen species. A wide variety of chemical and environmental factors are known to induce these antioxidant enzymes. Here, we examined the effect of ginseng saponins on the induction of SOD and catalase gene expression. To explore this possibility, the upstream regulatory promoter region of Cu/Zn superoxide dismutase (SOD1) and catalase genes were linked to the chloramphenicol acetyltransferase (CAT) structural gene and introduced into human hepatoma HepG2 cells. Total saponin and panaxatriol did not activate the transcription of SOD1 and catalase genes but panaxadiol increased the transcription of these genes about 2–3 fold. Among the panaxadiol ginsenosides, the Rb2 subfraction appeared to be a major inducer of SOD1 and catalase genes. The specificity of the Rb2 effect was further confirmed by time course‐ and dose‐dependent induction experiments. These results suggest that the panaxadiol fraction and its ginsenosides could induce the antioxidant enzymes which are important for maintaining cell viability by lowering the level of oxygen radical generated from intracellular metabolism. Copyright

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element.

Abstract The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals.

Extracellular alkaline proteases from alkalophilic Vibrio metschnikovii strain RH530

SummaryAlkaline proteases, named VapT and VapK, from Gram-negative alkalophilic Vibrio metschnikovii strain RH530 were purified and characterized. Both enzymes had optimum pH and temperature of 10. 5 and 60 °C, respectively. VapT and VapK retained 40 % and 80 %, respectively, of their initial activities at pH 12 after 24-h incubation at 25 °C. The half-lives of VapT and VapK were 10 min and 24 min, respectively, at pH 8 and 60 °C. Addition of Ca2+ extended their half-lives more than 20 fold. VapT and VapK retained over 30 % and 90 %, respectively, of their activities in the presence of 5 % SDS and 8 M urea. Analysis of amino acid composition showed that VapT contained seven cysteine residues and VapK did two. The N-terminal amino acid sequences of the proteases were determined and compared with those of Bacillus licheniformis subtilisin Carlsberg, Vibrio alginolyticus exoprotease A, and Tritirachium album proteinase K.

Spl and C/EBP-related factor regulate the transcription of human Cu/Zn SOD gene

The Cu/Zn superoxide dismutase (SOD1) is one of the key enzymes that protect cells against oxidative stress. It catalyzes the dismutation of superoxide radicals (O2-) to oxygen and hydrogen peroxide. To study the transcriptional regulation of the human Cu/Zn SOD gene, we began by analyzing the 1.5-kb upstream region of the gene (see Kim et al., 1994). The element from nucleotides (nt) -116 to -45 increased the transcriptional activity of Cu/Zn SOD. Analyses by DNase I footprinting and electrophoretic mobility shift assay (EMSA) showed that Sp1 binds to the region from nt -104 to -89 and C/EBP-related factors to the region from nt -64 to -55. Studies using two mutant versions of this promoter, in which the Sp1 and C/EBP-related factor binding sites were deleted, respectively, revealed that Sp1 and C/EBP-related factors activate the transcription of SOD1 gene. An Sp1 expression plasmid, pPacSp1, stimulated the SOD1-linked CAT expression. Cotransfection of the element from nt -116 to -45 with the C/EBP alpha expression vector, pMSV-C/EBP, increased the transcriptional activity of the Cu/Zn SOD in HepG2 cells, but barely in HeLa cells. Because Sp1 is a ubiquitously expressed transcriptional factor, the binding of Sp1 to the proximal upstream region of the Cu/Zn SOD might explain the expression of Cu/Zn SOD in a wide variety of cells.

Host cell proteins binding to the encapsidation signal epsilon in hepatitis B virus RNA.

Summary. The highly conserved encapsidation signal (ɛ) of hepatitis B viral (HBV) pregenomic RNA has been reported as an essential component for encapsidation and protein priming of HBV polymerase. Here, we report that two HBV ɛ RNA-binding host proteins (80 and 43 kDa) and a copurifying protein (100 kDa) were purified and characterized by the combined methods of UV cross-linking analysis with the ɛ RNA and column chromatography. Amino-terminal micro-sequencing showed that 80- and 43-kDa proteins were identified as the heterodimeric nuclear factor of activated T cells (NF90/NF45) and 100 kDa as a molecular chaperone, the GRP94. The heterodimeric factor interacted preferentially with the upper-bulge region of HBV ɛ RNA helping the HBV polymerase bind the lower-bulge region. Using in vitro protein priming analysis, the initial oligonucleotide of the protein-priming product was deduced as 5′-GAAC-3′, which is the complementary sequence of both regions of DR1 and ɛ in the pregenomic RNA. Previously, we also proposed that the GRP94 was associated with HBV polymerase in the human liver cell HepG2. These results suggest that the heterodimeric factor plays an important role in the priming activity of HBV polymerase.