Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Retinol stimulates the formation of transition vesicles in situ and in all free systems based on rat liver. The stimulation is on vesicle formation from transitional endoplasmic reticulum and not on vesicle fusion with donor membranes. Vesicle budding in the cell free system requires a nucleoside triphosphate and is sensitive to inhibition by thiol reagents. In this report we develop and test a model whereby a retinol-modulated NADH:protein disulfide reductase (NADH oxidase) with protein disulfide-thiol interchange activity is implicated in the vesicle budding mechanism. The protein has the ability to restore activity to scrambled, inactive RNase A and is stimulated or inhibited by retinol depending on the redox environment. Under reducing conditions and in the presence of a chemical reductant such as GSH, the partial reaction stimulated by retinol appears to be the oxidation of membrane thiols. This is the first report of an enzymatic mechanism to explain specific retinol effects both in vivo and in vitro on membrane trafficking not given by retinoic acid.
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PMID:A molecular basis for retinol stimulation of vesicle budding in vivo and in vitro. 978 45

We have examined the activity of protein disulfide isomerase (PDI) and glutaredoxin (Grx) 1, 2 and 3 from Escherichia coli to catalyze the cleavage of glutathionylated ribonuclease A (RNase-SG) by 1 mM GSH to yield reduced RNase. Apparent Km values for RNase-SG were similar, 2-10 microM, for Grx 1, 3 and PDI but Grx I and Grx 3 showed 500-fold higher turnover numbers than PDI. The atypical Grx 2 also catalyzed deglutathionylation by GSH, but had higher Km and apparent turnover number values compared to the two classical Grx. Refolding of RNase in a glutathione redox buffer was catalyzed by PDI. However, it could be measured only after a characteristic lag phase that was shortened by all three E. coli Grxs in a concentration-dependent manner. A role of the glutaredoxin mechanism in the endoplasmic reticulum is suggested.
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PMID:Reactivity of glutaredoxins 1, 2 and 3 from Escherichia coli and protein disulfide isomerase towards glutathionyl-mixed disulfides in ribonuclease A. 998 80

Angiogenins are proteins in the pancreatic ribonuclease superfamily that utilize their ribonuclease activity to induce formation of new blood vessels. Recently we identified a new member of the angiogenin gene family, mouse angiogenin-3, by virtue of its transcriptional activation in NIH3T3 fibroblasts coincident with transformation by the chimeric leukemia oncogene, E2a-Pbx1. Here we have isolated the cDNA encoding mouse angiogenin-3 and used it to produce the protein in E. coli. We demonstrate that mouse angiogenin-3 is a ribonuclease whose activity and specificity towards tRNA and dinucleotide substrates differ from those of mouse angiogenin or of mouse angiogenin-related protein, a non-angiogenic factor. Mouse angiogenin-3 induced angiogenesis in both the chicken embryo chorioallantoic membrane assay and the rat cremaster muscle. Electron microscopy revealed that endothelial cells within vessels induced by both mouse angiogenin-3 and mouse angiogenin contain fenestrations similar to those observed in endothelial cells from neovasculature induced by vascular endothelial growth factor and basic fibroblast growth factor. Mouse angiogenin-3 also induced other molecular events typical of rapidly proliferating endothelial cells, such as increases in rough endoplasmic reticulum, polysomes, and mitochondria.
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PMID:mAngiogenin-3, a target gene of oncoprotein E2a-Pbx1, encodes a new angiogenic member of the angiogenin family. 1059 12

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.
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PMID:Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. 1065 50


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