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)

UDP-glucose:glycoprotein glucosyltransferase (GT) is a key component of the glycoprotein-specific folding and quality control system in the endoplasmic reticulum. By exclusively reglucosylating incompletely folded and assembled glycoproteins, it serves as a folding sensor that prolongs the association of newly synthesized glycoproteins with the chaperone-like lectins calnexin and calreticulin. Here, we address the mechanism by which GT recognizes and labels its substrates. Using an improved inhibitor assay based on soluble conformers of pancreatic ribonuclease in its glycosylated (RNase B) and unglycosylated (RNase A) forms, we found that the protein moiety of a misfolded conformer alone is sufficient for specific recognition by GT in vitro. To investigate the relationship between recognition and glucosylation, we tested a variety of glycosylation mutants of RNase S-Protein and an RNase mutant with a local folding defect [RNase C65S, C72S], as well as a series of loop insertion mutants. The results indicated that local folding defects in an otherwise correctly folded domain could be recognized by GT. Only glycans attached to the polypeptide within the misfolded sites were glucosylated.
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PMID:Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT. 1586 Nov 39

We previously reported a genetic analysis of the growth-inhibitory effect caused by the overexpression of the Aspergillus oryzae rntA gene, encoding RNase T1 (Ribonuclease T1), in Saccharomyces cerevisiae. Subsequently, rns (ribonuclease T1 sensitive) mutants with mutations in the rns1 (DSL1), rns2 (UMP1), and rns3 (SEC17) genes, were identified. In the present study, rns4 (VPS32/SNF7) gene mutation was identified by complementation of tunicamycin sensitivity. While the rns4 mutant exhibited sensitivity to ambient stress conditions (200 mM CaCl(2), 1M NaCl and pH 8.0), genome-wide expression analysis revealed a similar pattern of genes up-regulated as was observed under nitrogen depletion condition by Gasch et al. [Mol. Biol. Cell 11 (2000) 4241]. Notably, the genes participating in autophagy (ATG4 and ATG8), the genes encoding a vacuolar protease (PRB1), vacuolar protease inhibitors (PAI3, PBI2 and TFS1) and YHR138c (a PBI2 homolog) were up-regulated in the rns4 mutant. Interestingly, the RNase T1*-GFP fusion protein (*inactive form) expressed in the rns4 mutant strain localized at the ER and vacuole under both stress or no-stress conditions. In contrast, the RNase T1*-GFP fusion protein expressed in the wild-type strain could not be detected under no-stress conditions, however, a stress-dependent localization of the fusion protein was observed at the vacuole. Since, the rns4 mutant exhibited a partial starvation-like response in spite of a rich ambient environment, leading to transportation of the secretory protein to the vacuole and accumulation in the endoplasmic reticulum, the present findings implicate a novel role for Rns4/Vps32 in proper response and adaptation to ambient conditions.
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PMID:Identification and characterization of rns4/vps32 mutation in the RNase T1 expression-sensitive strain of Saccharomyces cerevisiae: Evidence for altered ambient response resulting in transportation of the secretory protein to vacuoles. 1592 8

Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-A crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by alpha-helices that are situated on a beta-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.
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PMID:On the mechanism of sensing unfolded protein in the endoplasmic reticulum. 1636 12

ER-60 is a PDI family protein that has protein thiol-disulfide oxidoreductase activity. It has been shown to associate with BiP in the endoplasmic reticulum. Here, we analyzed the cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. ER-60 facilitated the refolding of 20 or 30% of denatured alpha-lactalbumin or ribonuclease B during incubation for 80 min, and these levels of nearly doubled on the addition of BiP to the reaction mixture. BiP alone could not refold denatured ribonuclease B, but could refold denatured alpha-lactalbumin a little. Enhancement of oxidative refolding of alpha-lactalbumin by ER-60 could be detected only when ER-60 was present from the start of refolding. On surface plasmon resonance analysis, ER-60 was shown to associate with BiP. The association was not influenced by ATP or ADP. Domains a and/or b' of ER-60 were implicated in the association with BiP.
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PMID:Cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. 1642 6

The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.
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PMID:The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH. 1666 42

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.
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PMID:Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION. 1666 93

The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.
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PMID:Crystal structure of the bb' domains of the protein disulfide isomerase ERp57. 1690 7

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.
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PMID:Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction. 1712 79

Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradation of misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol. Therefore, the enzyme was supposed to be able to distinguish between native and non-native glycoproteins. In the present study, a recombinant, yeast peptide:N-glycanase, Png1p, was expressed in Escherichia coli as inclusion bodies and was purified, refolded and characterized. The results showed that the recombinant enzyme has a broad pH range adaptation, from pH 4.0 to pH 10.0, and has an optimum temperature of 30 degrees C. This enzyme is a zinc metalloenzyme. Its activity was abolished with the addition of EDTA and not restored by adding metal ions. Furthermore, the deglycosylation efficiency of recombinant Png1p from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B (RNase B) was denatured at 60-65 degrees C or by 40-60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Png1p was most prominent. The deglycosylation efficiency of RNase B by Png1p was found to be related to its structural conformation and enzymatic activity.
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PMID:Influence of substrate conformation on the deglycosylation of ribonuclease B by recombinant yeast peptide:N-glycanase. 1721 53

The human signal recognition particle (SRP) is a large RNA-protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19-SRP54M-RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.
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PMID:Protein-induced conformational changes of RNA during the assembly of human signal recognition particle. 1725


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