Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ceruloplasmin (Cp) is a glycoprotein secreted by the liver and monocytic cells and probably plays roles in inflammation and iron metabolism. We showed previously that gamma interferon (IFN-gamma) induced Cp synthesis by human U937 monocytic cells but that the synthesis was subsequently halted by a transcript-specific translational silencing mechanism involving the binding of a cytosolic factor(s) to the Cp mRNA 3' untranslated region (UTR). To investigate how protein interactions at the Cp 3'-UTR inhibit translation initiation at the distant 5' end, we considered the "closed-loop" model of mRNA translation. In this model, the transcript termini are brought together by interactions of poly(A)-binding protein (PABP) with both the poly(A) tail and initiation factor eIF4G. The effect of these elements on Cp translational control was tested using chimeric reporter transcripts in rabbit reticulocyte lysates. The requirement for poly(A) was shown since the cytosolic inhibitor from IFN-gamma-treated cells minimally inhibited the translation of a luciferase reporter upstream of the Cp 3'-UTR but almost completely blocked the translation of a transcript containing a poly(A) tail. Likewise, a requirement for poly(A) was shown for silencing of endogenous Cp mRNA. We considered the possibility that the cytosolic inhibitor blocked the interaction of PABP with the poly(A) tail or with eIF4G. We found that neither of these interactions were inhibited, as shown by immunoprecipitation of PABP followed by quantitation of the poly(A) tail by reverse transcription-PCR and of eIF4G by immunoblot analysis. We considered the alternate possibility that these interactions were required for translational silencing. When PABP was depleted from the reticulocyte lysate with anti-human PABP antibody, the cytosolic factor did not inhibit translation of the chimeric reporter, thus showing the requirement for PABP. Similarly, in lysates treated with anti-human eIF4G antibody, the cytosolic extract did not inhibit the translation of the chimeric reporter, thereby showing a requirement for eIF4G. These data show that translational silencing of Cp requires interactions of three essential elements of mRNA circularization, poly(A), PABP, and eIF4G. We suggest that Cp mRNA circularization brings the cytosolic Cp 3'-UTR-binding factor into the proximity of the translation initiation site, where it silences translation by an undetermined mechanism. These results suggest that in addition to its important function in increasing the efficiency of translation, transcript circularization may serve as an essential structural determinant for transcript-specific translational control.
 ...
PMID:Translational silencing of ceruloplasmin requires the essential elements of mRNA circularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiation factor 4G. 1153 33

MagA is an iron-translocating protein found in the membranes of magnetic bacterium. Luciferase-bacterial magnetic particle (BMP) complexes were prepared by artificially inserting MagA-luciferase fusion proteins into the membranes of BMPs from Magnetospirillum magneticum strain AMB-1. Fusion proteins were from recombinant Escherichia coli membranes. MagA-Luc fusion proteins were integrated by sonication in vitro. Successful integration of fusion proteins was confirmed by luciferase luminescence on BMPs. Maximum luminescence was obtained after sonication for 3 min with a solution containing 300 mM NaCl, and is 18 times higher compared with recombinant Luc-BMPs generated using previously reported gene fusion techniques.
 ...
PMID:Preparation of luciferase-bacterial magnetic particle complex by artificial integration of MagA-luciferase fusion protein into the bacterial magnetic particle membrane. 1180 56

Insulin is a potent negative regulator of the response of hepatic cells to pro-inflammatory cytokines, particularly, interleukin (IL)-6. The action of insulin is target-selective because it inhibits transcription of most but not all acute phase genes. We here show that ceruloplasmin (Cp), an acute phase reactant with important functions in iron homeostasis, is subject to a unique dual regulation by insulin. IL-6 increased Cp mRNA expression in HepG2 cells by approximately 5-fold. Simultaneous treatment with insulin reduced this stimulation by half. Surprisingly, insulin by itself caused a 2-4-fold induction in Cp mRNA expression. The mechanism of induction by insulin was studied by transfecting into HepG2 cells chimeric constructs of the Cp 5'-flanking region driving luciferase. The activity of a 4800-bp segment of the Cp 5'-flanking region was increased 3-fold by insulin. Deletion and mutation analyses showed the requirement for a single hypoxia-responsive element in a 96-bp segment approximately 3600 bp upstream of the initiation site. The domains required for the two activities of insulin were distinct: The distal, hypoxia-responsive element-containing site was sufficient for Cp transactivation by insulin; in contrast, an 848-bp region adjacent to the initiation site was sufficient for IL-6 transactivation of Cp and for the inhibitory activity of insulin. The role of hypoxia-inducible factor-1 in the induction of Cp by insulin was shown by electrophoretic mobility shift assays and by the absence of insulin-stimulated Cp promoter activation in mouse Hepa c4 cells deficient in hypoxia-inducible factor-1 activity. Taken together these results show that insulin functions as a bidirectional, condition-dependent regulator of hepatic cell Cp expression. The unique regulation of Cp may reflect its dual roles in inflammation and iron homeostasis.
 ...
PMID:Dual role of insulin in transcriptional regulation of the acute phase reactant ceruloplasmin. 1202 93

Heme oxygenase is a rate-limiting enzyme in heme degradation, leading to the generation of free iron, biliverdin, and carbon monoxide. Induction of heme oxygenase-1 is implicated in the antioxidant defense mechanism and can modulate vascular function. To test the association of microsatellite polymorphism in the promoter region of human HO-1 gene with the risk of coronary artery disease (CAD) in type 2 diabetic patients, we examined the allele frequencies of (GT) (n) repeats in HO-1 gene in 474 patients with CAD and in 322 controls. A transient-transfection assay with HO-1 promoter/luciferase fusion constructs carrying various lengths of (GT) (n) repeats was performed to explore the regulatory effect of (GT) (n) repeats on HO-1 gene expression in cultured rat aortic smooth muscle cells. Serum thiobarbituric acid-reactive substances (TBARs), a measure of lipid peroxidation, was significantly higher in subjects carrying the L/L genotype (> or =32 repeats). Among type 2 diabetic subjects, the frequencies of the L alleles and proportion of genotypes with L alleles were significantly higher in those with CAD than in those without CAD. The adjusted odds ratio for CAD in type 2 diabetic patients with L alleles was 4.7 (95% confidence interval, 1.9-12.0, P=0.001). Transfection experiments in aortic smooth muscle cells revealed that HO-1 promoter/luciferase fusion constructs containing longer (GT) (n) repeats exhibited lower transcriptional activity. These results imply that the length polymorphism in the HO-1 gene promoter modulate the transcription of the gene in vascular cells. Type 2 diabetics carrying longer (GT) (n) repeats might have higher oxidative stress and increased susceptibility to the development of CAD.
 ...
PMID:Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. 1213 29

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor kappaB (NF-kappaB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-kappaB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-kappaB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H(2)O(2), significantly decreased DOX-induced NF-kappaB activation and apoptosis. Inhibition of DOX-induced NF-kappaB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-kappaB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IkappaB mutant vector, another NF-kappaB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-kappaB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-kappaB activation is anti-apoptotic. Removal of intracellular H(2)O(2) protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-kappaB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.
 ...
PMID:Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide. 1213 90

Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.
 ...
PMID:C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. Cross-talk between C/EBP pathway and iron metabolism. 1218 49

The eukaryotic Y-box-binding protein YB-1 functions in various biological processes, including DNA repair, cell proliferation, and transcriptional and translational controls. To gain further insight into how human YB-1 plays its role in pleiotropic functions, we here used two-hybrid screenings to identify partners of this protein; the results showed that YB-1 itself, iron-regulatory protein 2 (IRP2), and five ribosomal proteins each served as partners to YB-1. We then examined the biological effect of the interaction of YB-1 and IRP2 on translational regulation. Both in vitro binding and coimmunoprecipitation assays showed the direct interaction of YB-1 and IRP2 in the presence of a high concentration of iron. RNA gel shift assays showed that YB-1 reduced the formation of the IRP2-mRNA complex when the iron-responsive element of the ferritin mRNA 5' untranslated region (UTR) was used as a probe. By using an in vitro translation assay using luciferase mRNA ligated to the ferritin mRNA 5'UTR as a reporter construct, we showed that both YB-1 and IRP2 inhibited the translation of the mRNA. However, coadministration of YB-1 and IRP2 proteins abrogated the inhibition of protein synthesis by each protein. An In vivo coimmunoprecipitation assay showed that IRP2 bound to YB-1 in the presence of iron and a proteasome inhibitor. The direct interaction of YB-1 and IRP2 provides the first evidence of the involvement of YB-1 in the translational regulation of an iron-related protein.
 ...
PMID:Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2. 1219 37

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5'-untranslated region (5'-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (+51 to +94 from the 5'-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). APP 5'-UTR conferred translation was selectively down-regulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5'-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5'-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (Delta4 bases:Delta83AGAG86). Iron regulation of APP mRNA through the APP 5'-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce Abeta peptide burden during Alzheimer's disease.
 ...
PMID:An iron-responsive element type II in the 5'-untranslated region of the Alzheimer's amyloid precursor protein transcript. 1219 35

The erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis. The hypoxia-inducible factor-1 (HIF-1) transcriptionally up-regulates erythropoietin, transferrin, and transferrin receptor, leading to increased erythropoiesis and hematopoietic iron supply. To test the hypothesis that ALAS2 expression might be regulated by a similar mechanism, we exposed murine erythroleukemia cells to hypoxia (1% O(2)) and found an up to 3-fold up-regulation of ALAS2 mRNA levels and an increase in cellular heme content. A fragment of the ALAS2 promoter ranging from -716 to +1 conveyed hypoxia responsiveness to a heterologous luciferase reporter gene construct in transiently transfected HeLa cells. In contrast, iron depletion, known to induce HIF-1 activity but inhibit ALAS2 translation, did not increase ALAS2 promoter activity. Mutation of a previously predicted HIF-1-binding site (-323/-318) within this promoter fragment and DNA-binding assays revealed that hypoxic up-regulation is independent of this putative HIF-1 DNA-binding site.
 ...
PMID:Hypoxic up-regulation of erythroid 5-aminolevulinate synthase. 1239 45

Previous studies have shown cytochrome P450 2E1 (CYP2E1)-dependent transcriptional up-regulation of glutamate-cysteine ligase (GCL). To identify sequences mediating constitutive and induced expression of the catalytic subunit of GCL (GCLC), a series of deletion mutants from the 5'-flanking region (-3,802 to +465) were transfected into control (C34) and CYP2E1-overexpressing (E47) HepG2 cells. Increased luciferase expression, both basal (2- to 3-fold) and following exposure to ethanol, arachidonic acid (AA), or AA plus iron, was detected in E47 cells with the full-length but not shorter reporter vectors. Basal induction was blocked by CYP2E1 inhibitors and catalase. Basal and inducible luciferase expression in E47 cells was blunted by the full-length construct mutated in the ARE4 site. Catalase and diallyl sulfide prevented basal and AA-induced messenger RNA (mRNA) levels of GCLC and the modulatory subunit of GCL (GCLM). Preincubation with low doses of AA increased glutathione (GSH) levels as well as GCLC and GCLM mRNAs, and this protected against H(2)O(2) and menadione toxicity. Primary hepatocytes from pyrazole-injected rats with high levels of CYP2E1 showed an increase in GSH levels as well as GCLC and GCLM mRNAs compared with saline controls, and this was prevented by diallyl sulfide. In conclusion, redox-sensitive elements directing constitutive and induced expression of the GCLC in CYP2E1-expressing cells are present in the ARE4 distal portion of the 5'-flanking region, between positions -3,802 and -2,752, perhaps a reflection of metabolic adaptation to CYP2E1-generated oxidative stress.
 ...
PMID:Cytochrome P450 2E1 responsiveness in the promoter of glutamate-cysteine ligase catalytic subunit. 1250 Jan 94


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>