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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 A resolution to a conventional R-factor ( = sigma magnitute of (magnitute of F(zero)-magnitude of Fc)/ sigma magnitude of F(zero) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO4(B) binding site of rEDN likely corresponds to the P-1 subsite and may be representative of how other homologous RNases bind the P-1 phosphate.
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PMID:X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83 A resolution. 875 19

The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our ongoing efforts to trace the evolutionary history of the ribonuclease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino acid polypeptide with eight cysteines and histidine and lysine residues corresponding to those found in the active site of the prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous sequences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human tissues tested, with lung representing the most abundant source. At the cellular level, transcripts encoding RNase k6 were detected in normal human monocytes and neutrophils (but not in eosinophils) suggesting a role for this ribonuclease in host defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence identity; slight cross-reactivity between RNase k6 and EDN was observed on Western blots probed with polyclonal anti-EDN antiserum. The catalytic constants determined, Km = 5.0 microM and Kcat = 0.13 s-1, indicate that recombinant RNase k6 has approximately 40-fold less ribonuclease activity than recombinant EDN. The identification and characterization of RNase k6 has extended the ribonuclease gene family and suggests the possibility that there are others awaiting discovery.
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PMID:Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family. 883 75

The eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are both small, cationic ribonuclease toxins that are stored in and secreted by activated human eosinophilic leukocytes. We have previously shown that optimal expression of the EDN gene is dependent on an interaction between an intronic enhancer element or elements and the 5' promoter region. Here we present evidence demonstrating that the gene encoding ECP is regulated in an analogous fashion and that an intronic enhancer element functioning in both genes is a consensus binding sequence for the transcription factor NFAT-1. Our initial results demonstrate that one or more nuclear proteins isolated from human promyelocytic leukemia (HL-60) cells bind specifically at this consensus site (5'-GGAGAA-3') within the intron of the EDN gene and that disruption of this sequence reduced the characteristic 20-30-fold increase in reporter gene activity observed with the tandem EDN promoter/exon 1/intron construct to background levels. The NFAT-1 consensus site in the ECP gene differs from that found in the EDN gene by a single nucleotide (5'-GGAGAG-3'); the conversion of the 3' G to an A resulted in a further enhancement of the reporter gene activity supported by the ECP promoter/exon 1/intron construct. Interestingly, no "supershift" was observed in gel shift assays performed in the presence of anti-NFAT-1 antiserum, suggesting that a nuclear protein other than NFAT-1 may be acting at this consensus site.
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PMID:Intronic enhancer activity of the eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3) genes is mediated by an NFAT-1 consensus binding sequence. 899 43

Using the rapid amplification of cDNA ends (RACE) procedure, we have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil cationic protein (ECP)/eosinophil-associated ribonuclease (EAR). The deduced amino acid sequence revealed that the molecular weight of rat preECP/EAR is 18.0 kDa and the isoelectric point is 9.85, indicating that rat ECP/EAR is highly cationic. The homology of amino acid sequence between rat ECP/EAR and human ECP is 54%, and that between rat ECP/EAR and human eosinophil-derived neurotoxin (EDN) is 51%. Rat ECP/EAR is also homologous to human ribonuclease k6 (homology 47%).
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PMID:Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease. 911 43

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.
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PMID:Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy. 919 72

The human eosinophil-derived neurotoxin (hEDN) is a secretory effector protein from eosinophilic leukocytes that is a member of the ribonuclease A (RNase A) family of ribonucleases. EDN is a rapidly evolving protein, accumulating non-silent mutations at a rate exceeding those of most other functional coding sequences studied in primates. Although all primate EDNs retain the structural and functional residues known to be prerequisites for ribonuclease activity, we have shown previously that recombinant EDN derived from a New World monkey sequence ( Saguinus oedipus ) had significantly less catalytic activity than the human (hEDN) ortholog.In this work, we have prepared recombinant proteins from EDN from sequences derived from orangutan (Pongo pygmaeus, oEDN) and Old World monkey (Macaca fascicularis, mcEDN) genomic DNAs, and from a second New World monkey sequence (Aotus trivirgatus, omEDN) as well. The catalytic efficiencies [ k cat/ K m (M-1s-1)] determined for both oEDN and mcEDN were similar to that determined previously for hEDN, while omEDN displayed approximately 100-fold less catalytic activity. The relative ribonuclease activities of hEDN/omEDN chimeras pointed to a C-terminal segment as crucial to the enhanced catalytic activity hEDN, and substitution of Arg 132-Ile 133 of hEDN with the Thr-Thr pair at the analogous position in omEDN resulted in an approximately 10-fold reduction in hEDN's catalytic efficiency. However, the reverse substitution, Arg-Ile for Thr-Thr in omEDN, did not enhance the catalytic efficiency of this relatively inactive protein. These results indicate that the Arg and/or Ile residues adjacent to the C-terminus are necessary (but not sufficient) for enhanced ribonuclease activity among the primate EDNs, and will permit prediction of the relative ribonuclease activities based on differences in primary structure.
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PMID:Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity. 925 15

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.
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PMID:Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism. 930 75

Evolutionary mechanisms of origins of new gene function have been a subject of long-standing debate. Here we report a convincing case in which positive Darwinian selection operated at the molecular level during the evolution of novel function by gene duplication. The genes for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in primates belong to the ribonuclease gene family, and the ECP gene, whose product has an anti-pathogen function not displayed by EDN, was generated by duplication of the EDN gene about 31 million years ago. Using inferred nucleotide sequences of ancestral organisms, we showed that the rate of nonsynonymous nucleotide substitution was significantly higher than that of synonymous substitution for the ECP gene. This strongly suggests that positive Darwinian selection operated in the early stage of evolution of the ECP gene. It was also found that the number of arginine residues increased substantially in a short period of evolutionary time after gene duplication, and these amino acid changes probably produced the novel anti-pathogen function of ECP.
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PMID:Positive Darwinian selection after gene duplication in primate ribonuclease genes. 952 Apr 31

An HIV-1-based expression vector has been constructed that produces protective genes tightly regulated by HIV-1 Tat and Rev proteins. The vector contains either a single protective gene (HIV-1 gag dominant negative mutant (delta-gag)) or a combination of two different protective genes (delta-gag and eosinophil-derived neurotoxin (EDN), a human ribonuclease) which are expressed from a dicistronic mRNA. After stable transfection of CEM T cells and following challenge with HIV-1, viral production was completely inhibited in cells transduced with the vector producing both delta-gag and EDN and delayed in cells producing delta-gag alone. In addition, cotransfection of HeLa-Tat cells with an infectious HIV-1 molecular clone and either protective vector demonstrated that the HIV-1 packaging signals present in the constructs were functional and allowed the efficient assembly of the protective RNAs into HIV-1 virions, thus potentially transmitting protection to the HIV-1 target cells.
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PMID:Inhibition of HIV-1 replication by combined expression of gag dominant negative mutant and a human ribonuclease in a tightly controlled HIV-1 inducible vector. 953 66

A dose-dependent decrease in infectivity was observed on introduction of eosinophils into suspensions of respiratory syncytial virus group B (RSV-B). This antiviral effect was reversed by ribonuclease inhibitor, suggesting a role for the eosinophil secretory ribonucleases. Recombinant eosinophil-derived neurotoxin (rhEDN), the major eosinophil ribonuclease, promoted a dose-dependent decrease in RSV-B infectivity, with a 40-fold reduction observed in response to 50 nM rhEDN. Ribonucleolytically inactivated rhEDN (rhEDNdK38) had no antiviral activity. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated loss of viral genomic RNA in response to rhEDN, suggesting that this protein promotes the direct ribonucleolytic destruction of extracellular virions. Ribonuclease A had no antiviral activity even at approximately 1000-fold higher concentrations, suggesting that rhEDN has unique features other than ribonuclease activity that are crucial to its effectiveness. These results suggest that rhEDN may have potential as a therapeutic agent for prevention or treatment of disease caused by RSV.
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PMID:Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus. 960 20


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