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)

Bovine seminal RNase (BS-RNase) is a dimeric RNase selectively cytotoxic for malignant cells. No information is available on its pathway from the extracellular matrix through the cytosol, where it degrades rRNA. An investigation of this pathway is reported here, carried out by immunofluorescence studies, by assessing the effects on BS-RNase cytotoxicity of drugs that affect specific intracellular compartments and by assaying the behaviour of a protein variant, BS-RNase-KDEL (BS-RNase in which a Lys-Asp-Glu-Leu peptide segment is inserted at the C-terminal ends of the subunit chains), endowed with a consensus sequence that directs proteins to the endoplasmic reticulum. BS-RNase was found to bind both normal and malignant cells and to be internalized by both cell types in endosome vesicles. Non-cytotoxic RNases, such as RNase A and a monomeric derivative of BS-RNase, did not bind to the cell surface and were not internalized. However, an engineered, dimeric and cytotoxic variant of RNase A bound effectively and permeated cells. The results of immunofluorescence studies, the effects of nigericin, monensin and brefeldin A on the cytotoxic action of seminal RNase, and the behaviour of the BS-RNase-KDEL variant, led to the conclusion that the pathway of BS-RNase in malignant cells from the extracellular matrix to the cytosol has two essential intracellular stations: endosomes and the trans-Golgi network. In normal cells, however, the protein does not progress from the endosomal compartment to the Golgi complex.
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PMID:Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease. 1187 81

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.
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PMID:Isolation and characterization of a putative transducer of endoplasmic reticulum stress in Oryza sativa. 1204 Jan

BACE is the beta-secretase responsible for the first step in amyloidogenic processing of the amyloid precursor protein APP. We have identified two BACE isoforms, BACE1B and BACE1C, lacking 25 and 44 amino acids, respectively. Whereas the BACE1B transcript is present in human pancreas and brain, the BACE1C transcript is found in pancreas only. In transfected cells both BACE1A, which encodes the originally described full-length BACE1 protein and the close homolog BACE2 localized mainly to post-Golgi membranes. In contrast, the two shorter isoforms were found in the endoplasmic reticulum only, and they did not display beta-secretase activity. Using RNase protection we in addition show that the major pancreatic transcript is BACE1A. This suggests that the known absence of beta-secretase activity in the pancreas is not due to a missing BACE1A transcript.
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PMID:Splice variants of the beta-site APP-cleaving enzyme BACE1 in human brain and pancreas. 1205 59

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.
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PMID:Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor. 1214 88

Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is mediated by transport vesicles coated with the coat protein complex II (COPII). In the process of searching for novel factors that participate in the formation of COPII-coated vesicles (COPII vesicles), we isolated high-copy suppressors of a sec24-20 mutant defective in COPII vesicle formation from the ER at the restrictive temperature. Unexpectedly, one of them was identified as HAC1, a gene encoding the basic leucine-zipper type transcription factor Hac1p. Hac1p is essential for a signaling cascade activated by ER stress, termed the unfolded protein response (UPR) pathway, that leads from the ER to the nucleus. Overexpression of another UPR-related gene IRE1, which encodes an ER-resident transmembrane protein kinase/ribonuclease, also suppressed the growth defect of the sec24-20 mutant in a HAC1-dependent manner. Moreover, overexpression of IRE1 specifically suppressed growth defects of other sec mutants defective in COPII vesicle formation. These findings suggest that the activation of the UPR affects ER-to-Golgi transport via stimulation of COPII vesicle formation from the ER.
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PMID:A genetic link between the unfolded protein response and vesicle formation from the endoplasmic reticulum. 1217 18

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.
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PMID:Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes. 1217 51

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.
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PMID:Ribonuclease activity and RNA binding of recombinant human Dicer. 1241 4

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.
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PMID:Inducible system in human hepatoma cell lines for hepatitis C virus production. 1248 60

In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.
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PMID:Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. 1280 51

Conditions that perturb the function of the endoplasmic reticulum (ER) lead to an accumulation of proteins and subsequent induction of several responses, such as an increased expression of ER-resident chaperones involved in protein folding and activation of c-jun N-terminal kinase (JNK). These responses are mediated by a transmembrane kinase/ribonuclease, IRE1, which transduces the signal from the ER lumen to the cytosol. Although nuclear transcription factor-kappaB (NF-kappaB) is also activated by ER stress, whether this response depends on IRE1 is unknown. In this study, we show that IRE1 is involved in the activation of NF-kappaB induced by ER stress. NF-kappaB was activated by ER stress-inducing agents, thapsigargin and tunicamycin. The activation was inhibited by a dominant-negative IRE1. In addition, a dominant-negative TRAF2 also suppressed the activation of NF-kappaB by ER stress. These results suggest that ER stress-induced NF-kappaB activation is also mediated by the IRE1-TRAF2 pathway, as well as JNK activation.
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PMID:Activation signal of nuclear factor-kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. 1284 13


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