Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Parathyroid hormone-related protein (PTHrP) has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). The regulation and mechanisms of PTHrP secretion in most normal and malignant cells are unknown. PTHrP secretion, mRNA expression, and transcription were measured in neoplastic human squamous carcinoma cells (A253) and normal human foreskin keratinocytes (NHFK) by radioimmunoassay, RNase protection assay, and transient transfections of the 5'-flanking region of human PTHrP in a luciferase expression vector. Mechanisms of PTHrP secretion were investigated using chemicals (monensin, colchicine, cytochalasin B, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS)) that interfere with or facilitate intracellular transport. Monensin inhibited PTHrP secretion in both NHFK and A253 cells. Ultrastructurally, monensin caused dilatation of rough endoplasmic reticulum and the formation of numerous cytoplasmic secretory vacuoles in both cell lines. Colchicine decreased PTHrP production in NHFK cells and stimulated PTHrP production and mRNA levels in A253 cells. Colchicine also stimulated transcription of the PTHrP-luciferase reporter gene. Cytochalasin B stimulated PTHrP secretion and mRNA expression in A253 cells, but had no effect in NHFK cells. GTPgammaS had no effect on PTHrP secretion in either cell line. It was concluded that PTHrP secretion is dependent on the constitutive movement of secretory vesicles to the cytoplasmic membrane and regulation of PTHrP secretion and mRNA expression are altered in squamous carcinoma cells compared to normal human keratinocytes in vitro.
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PMID:Regulation of parathyroid hormone-related protein secretion and mRNA expression in normal human keratinocytes and a squamous carcinoma cell line. 914 24

Activation of the cellular immune system and subsequent lysis of vector-transduced cells by adenovirus- or transgene-specific cytotoxic T lymphocytes have been shown to limit transgene expression in animal models. The adenovirus gp19K gene product associates with major histocompatibility complex class I proteins and prevents their maturation by sequestering them in the endoplasmic reticulum. gp19K has been shown to block the ability of adenovirus-specific cytotoxic T lymphocytes to recognize virus-infected cells in vitro. To determine if gp19K expression in an adenovirus vector would increase transgene persistence, a vector that replaces the E1 region of adenovirus with an expression cassette encoding both gp19K and beta-glucuronidase was constructed. This vector produced high levels of functional gp19K in infected cells. RNase protection analysis revealed efficient expression of the gp19K gene in the mouse lung. Enhanced persistence and increased beta-glucuronidase activity were observed in the lung and liver following delivery of the gp19K-expressing adenovirus vector in B10.HTG mice but not in BALB/c mice. Since gp19K binds to both class I alleles on B10.HTG mice but only one allele on BALB/c mice, these results suggest that the major histocompatibility complex class I haplotype of mice is important in determining the effectiveness of gp19K in vivo. Since gp19K has previously been shown to interact with every human major histocompatibility complex class I allele tested, the inclusion of gp19K in gene therapy vectors may increase vector persistence in human gene therapy trials.
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PMID:Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. 931 44

We demonstrated that overexpression of a cRNA encoding a truncated potassium channel polypeptide that contains the NH2 terminus and the first transmembrane segment (Kv1.1N206Tag) abolished the expression of Kv1.1 and Kv1.5 outward currents in Xenopus oocytes (Babila, T., Moscucci, A., Wang, H., Weaver, F. E. & Koren, G. (1994) Neuron 12, 615-626). Recently, we showed that expression of Kv1.1N206Tag in the heart of transgenic mice resulted in the creation of mice with prolongation of the surface electrocardiogram's QT interval (London, B., Han, X., Folco, E. & Koren, G. (1996) Biophys. J. 70, A2601). To study the dominant negative mechanism of Kv1.1N206Tag, we overexpressed it in GH3 cells, a pituitary cell line expressing Kv1. 5 and Kv1.4. RNase protection analysis comparing the steady-state levels of native Kv1.5 and Kv1.1N206Tag transcripts revealed an excess of Kv1.1N206Tag transcript. Immunoprecipitation analysis using 12CA5 monoclonal antibody detected a 25-kDa polypeptide in the transfected cells. The half-life of Kv1.1N206Tag was 2.6 h. Subcellular fractionation of cell lysates labeled with [35S]methionine revealed that Kv1.1N206Tag polypeptide is detectable in the particulate (membranous) fraction, but not in the soluble (cytosol) fraction. A series of double immunoprecipitations with 12CA5 and polyclonal antibodies against Kv1.5 and Kv1.4 revealed that Kv1.1N206Tag forms heteromultimeric complexes with the native Kv1.4 and Kv1.5 polypeptides. The steady-state levels of Kv1.5 were not affected by the overexpression of Kv1.1N206Tag. Immunofluorescence colocalization and confocal microscopy analyses revealed that Kv1.1N206TagFlag did not reach the plasma membrane, and its distribution pattern was characteristic to that of a resident endoplasmic reticulum polypeptide. Our observations establish that the negative effect of Kv1.1N206Tag is mediated by the formation of heteromultimeric complexes with the native channels and by the retention of these complexes in the endoplasmic reticulum.
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PMID:A cellular model for long QT syndrome. Trapping of heteromultimeric complexes consisting of truncated Kv1.1 potassium channel polypeptides and native Kv1.4 and Kv1.5 channels in the endoplasmic reticulum. 933 28

Transglutaminases (TGases; EC 2.3.2.13) are a family of enzymes that catalyze calcium-dependent covalent cross-linking of cellular proteins by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds. These covalent isopeptide bonds are of great physiological significance because they are highly resistant to proteolysis, denaturants, and reducing agents. Prior studies have demonstrated the presence of isopeptide bonds in the sheath and cuticle of filarial parasites, suggesting an important role for TGase-catalyzed reactions during the growth and development of filarial nematodes. Herein we report the identification and cloning of a cDNA encoding a TGase from the dog heartworm Dirofilaria immitis (DiTG). The DiTG expressed in Escherichia coli (recombinant DiTG) was able to catalyze calcium-dependent cross-linking reactions. The derived amino acid sequence of the DiTG cDNA (pDiTG) predicts a protein of 57.1 kDa and includes an N-terminal hydrophobic signal peptide. The pDiTG has no sequence similarity with any of the known TGases, but it has significant homology to protein disulfide isomerase (PDI) and, particularly, to the PDI-related endoplasmic reticulum protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. As predicted from the amino acid sequence homology, recombinant DiTG catalyzed the isomerization of intramolecular disulfide/sulfhydryl bonds in denatured RNase in vitro as effectively as did mammalian PDI. Conversely, purified PDI from bovine liver could catalyze protein cross-linking reactions in a Ca(2+)-dependent manner. This report describes the dual catalytic activity of TGase and PDI in post- and/or cotranslational modification of newly synthesized proteins. These TGase-catalyzed posttranslational modifications may play a pivotal role in the synthesis of new cuticle during the growth and maturation of filarial parasites.
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PMID:An ERp60-like protein from the filarial parasite Dirofilaria immitis has both transglutaminase and protein disulfide isomerase activity. 943 26

Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.
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PMID:Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins. 948 8

The endoplasmic reticulum is the site of folding, disulfide bond formation, and N-glycosylation of secretory proteins. Correctly folded proteins are exported from the endoplasmic reticulum, whereas incorrectly folded proteins are retained by a quality control system. The type I membrane-protein calnexin and its soluble homologue calreticulin are constituents of this system that recognize monoglucosylated N-linked glycans that are present on unfolded glycoproteins. Although several components of the quality control apparatus are well characterized, it is not known whether and how they interact with enzymes that catalyze protein folding. The endoplasmic reticulum protein ERp57 is homologous to protein-disulfide isomerase and can be cross-linked to the same monoglucosylated glycoproteins that bind to calnexin and calreticulin. The present study demonstrates that the disulfide isomerase activity of ERp57 on the refolding of monoglucosylated ribonuclease B is much greater when this glycoprotein is associated with calnexin or calreticulin. This result is in contrast to protein-disulfide isomerase, whose activity on monoglucosylated ribonuclease B is decreased in the presence of these lectins. No direct binding of monoglucosylated ribonuclease B or monoglucosylated glycans to ERp57 could be detected, but we show that ERp57 interacts directly with calnexin.
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PMID:Enhanced catalysis of ribonuclease B folding by the interaction of calnexin or calreticulin with ERp57. 949 14

Unfolded proteins in the endoplasmic reticulum of Saccharomyces cerevisiae trigger a specialized RNA splicing event that allows the subsequent translation of the Hac1p transcription factor. This splicing can be reconstituted with Ire1p, a transmembrane kinase that has a site-specific RNase activity, and tRNA ligase.
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PMID:Splicing: HACking into the unfolded-protein response. 950 74

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting betaTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.
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PMID:In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation. 960 Sep 32

Our previous studies suggested that enhanced fibronectin mRNA translation in ductus arteriosus compared with aortic smooth muscle cells is related to increased expression of light chain 3 (LC3) of microtubule-associated protein 1, which binds an AU-rich element in the 3' untranslated region of fibronectin mRNA. We therefore hypothesized that microtubules are involved in LC3-mediated fibronectin mRNA translational regulation. In this study we show that disruption of microtubules by colchicine inhibits fibronectin mRNA translation in cultured ductus arteriosus smooth muscle cells. We proposed that the mechanism might be related to decreased docking of fibronectin mRNA on the translational machinery, ie, membrane-bound polysomes on rough endoplasmic reticulum, and confirmed this by Northern blot analysis. To investigate the mechanism further, we carried out polysome analysis using sucrose gradient centrifugation and fractionation and studied the polysomal distribution of fibronectin mRNA and LC3 protein in the sucrose gradient by using RNase protection assay and Western immunoblotting, respectively. Colchicine treatment shifts fibronectin mRNA from the fractions containing membrane-bound polysomes to the fractions carrying free polysomes and concomitantly decreases the amount of LC3 protein in the fractions containing membrane-bound polysomes. Furthermore, an EDTA-release experiment demonstrates that LC3 protein associates with the 60S ribosomal subunit. Our data support the concept that microtubules may function with LC3 to facilitate sorting of fibronectin mRNA onto rough endoplasmic reticulum and translation.
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PMID:Microtubule involvement in translational regulation of fibronectin expression by light chain 3 of microtubule-associated protein 1 in vascular smooth muscle cells. 973 70

The key feature of tomato RNase LX localised solely outside the vacuole is the C-terminal peptide HDEF which is very similar to known endoplasmic reticulum (ER) retention signals. For functional testing of the ER-targeting ability of HDEF, different constructs including the complete RNase LX, two truncated forms without HDEF and the truncated chitinase FB7-1deltaVTP C-terminally flanked by HDEF, were expressed in Saccharomyces cerevisiae. The majority of RNase and chitinase, both containing HDEF, accumulates within the ER. However, the truncated constructs without the peptide are released into the medium. We provide compelling evidence that peptide HDEF at the C-terminus of secretory plant proteins is a new ER retention signal in yeast and most likely in plants.
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PMID:The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum. 974 58


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