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)

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

We have localized the gene encoding human RNase k6 to within approximately 120 kb on the long (q) arm of chromosome 14 by HAPPY mapping. With this information, the relative positions of the six human RNase A ribonucleases that have been mapped to this locus can be inferred. To further our understanding of the individual lineages comprising the RNase A superfamily, we have isolated and characterized 10 novel genes orthologous to that encoding human RNase k6 from Great Ape, Old World, and New World monkey genomes. Each gene encodes a complete ORF with no less than 86% amino acid sequence identity to human RNase k6 with the eight cysteines and catalytic histidines (H15 and H123) and lysine (K38) typically observed among members of the RNase A superfamily. Interesting trends include an unusually low number of synonymous substitutions (Ks) observed among the New World monkey RNase k6 genes. When considering nonsilent mutations, RNase k6 is a relatively stable lineage, with a nonsynonymous substitution rate of 0.40 x 10(-9) nonsynonymous substitutions/nonsynonymous site/year (ns/ns/yr). These results stand in contrast to those determined for the primate orthologs of the two closely related ribonucleases, the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), which have incorporated nonsilent mutations at very rapid rates (1.9 x 10(-9) and 2.0 x 10(-9) ns/ns/yr, respectively). The uneventful trends observed for RNase k6 serve to spotlight the unique nature of EDN and ECP and the unusual evolutionary constraints to which these two ribonuclease genes must be responding. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF037081-AF037090.]
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PMID:Ribonuclease k6: chromosomal mapping and divergent rates of evolution within the RNase A gene superfamily. 964 35

Eosinophil cationic protein (ECP) is one of two RNase A-superfamily ribonucleases found in secretory granules of human eosinophilic leukocytes. Although the physiologic function of eosinophils [and thus of the two eosinophil ribonucleases, ECP and eosinophil-derived neurotoxin (EDN)] remains controversial, we have recently shown that isolated human eosinophils promote ribonuclease-dependent toxicity toward extracellular virions of the single-stranded RNA virus, respiratory syncytial virus, group B (RSV-B). We have also shown that recombinant human EDN (rhEDN) can act alone as a ribonuclease-dependent antiviral agent. In this work, we provide a biochemical characterization of recombinant human ECP (rhECP) prepared in baculovirus, and demonstrate that rhECP also promotes ribonuclease-dependent antiviral activity. The rhECP described here is N-glycosylated, as is native ECP, and has approximately 100-fold more ribonuclease activity than non-glycosylated rhECP prepared in bacteria. The enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor (RI). Although rhECP was not as effective as rhEDN at reducing viral infectivity (500 nM rhECP reduced infectivity of RSV-B approximately 6 fold; 500 nM rhEDN, >50 fold), the antiviral activity appears to be unique to the eosinophil ribonucleases; no reduction in infectivity was promoted by bovine RNase A, by the amphibian ribonuclease, onconase, nor by the closely-related human ribonuclease, RNase k6. Interestingly, combinations of rhEDN and rhECP did not result in either a synergistic or even an additive antiviral effect. Taken together, these results suggest that that the interaction between the eosinophil ribonucleases and the extracellular virions of RSV-B may be specific and saturable.
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PMID:Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. 964 19

Telomere maintenance executed by the action of telomerase seems to be a prerequisite for immortalization. Telomerase is found in most cell lines and malignant tumors. A telomerase-independent mechanism for telomere maintenance in Hodgkin's disease has been proposed in the absence of detectable telomerase activity. In this study, telomerase activity was detected in 31 of 77 Hodgkin's disease samples and a strong correlation between eosinophilia and absence of detectable telomerase activity was found. Purified eosinophils and specifically eosinophil-derived neurotoxin and eosinophilic cationic protein, both ribonucleases, were found to degrade telomerase. Purified neutrophils also exhibited weak telomerase degradative activity. Reanalysis of previously telomerase-negative Hodgkin's disease samples with eosinophilia using ribonuclease inhibitors resulted in the detection of telomerase activity. Ribonuclease-containing cells in vivo thus have a considerable impact on the detectability of telomerase. In Hodgkin's disease samples without eosinophilia, 24 of 27 exhibited telomerase activity at decreased levels compared with non-Hodgkin's lymphomas and at increased levels compared with reactive nodes indicative of a telomerase positive tumor component in Hodgkin's disease. Telomerase positivity of the Hodgkin's and Reed-Sternberg cells in vivo was also supported by high levels of telomerase expression in Hodgkin's disease cell lines. Based on our data, Hodgkin's lymphomas are potential targets for antitelomerase therapy.
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PMID:Telomerase activity in Hodgkin's disease. 965 57

We have demonstrated that the human eosinophil-derived neurotoxin (EDN, RNase 2), a rapidly evolving secretory protein derived from eosinophilic leukocytes, mediates the ribonucleolytic destruction of extracellular virions of the single-stranded RNA virus respiratory syncytial virus (RSV). While RNase activity is crucial to antiviral activity, it is clearly not sufficient, as our results suggest that EDN has unique structural features apart from RNase activity that are necessary to promote antiviral activity. We demonstrate here that the interaction between EDN and extracellular virions of RSV is both saturatable and specific. Increasing concentrations of the antivirally inactivated, ribonucleolytically inactivated point mutant form of recombinant human EDN, rhEDNdK38, inhibits rhEDN's antiviral activity, while increasing concentrations of the related RNase, recombinant human RNase k6, have no effect whatsoever. Interestingly, acquisition of antiviral activity parallels the evolutionary development of the primate EDN lineage, having emerged some time after the divergence of the Old World from the New World monkeys. Using this information, we created ribonucleolytically active chimeras of human and New World monkey orthologs of EDN and, by evaluating their antiviral activity, we have identified an N-terminal segment of human EDN that contains one or more of the sequence elements that mediate its specific interaction with RSV.
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PMID:Evolution of antiviral activity in the ribonuclease A gene superfamily: evidence for a specific interaction between eosinophil-derived neurotoxin (EDN/RNase 2) and respiratory syncytial virus. 982 55

Human angiogenin (Ang), an unusual member of the pancreatic RNase superfamily, is a potent inducer of angiogenesis in vivo. Its ribonucleolytic activity is weak (10(4) to 10(6)-fold lower than that of bovine RNase A), but nonetheless seems to be essential for biological function. Ang has been implicated in the establishment of a wide range of human tumours and has therefore emerged as an important target for the design of new anti-cancer compounds. We report high-resolution crystal structures for native Ang in two different forms (Pyr1 at 1.8 A and Met-1 at 2.0 A resolution) and for two active-site variants, K40Q and H13A, at 2.0 A resolution. The native structures, together with earlier mutational and biochemical data, provide a basis for understanding the unique functional properties of this molecule. The major structural features that underlie the weakness of angiogenin's RNase activity include: (i) the obstruction of the pyrimidine-binding site by Gln117; (ii) the existence of a hydrogen bond between Thr44 and Thr80 that further suppresses the effectiveness of the pyrimidine site; (iii) the absence of a counterpart for the His119-Asp121 hydrogen bond that potentiates catalysis in RNase A (the corresponding aspartate in Ang, Asp116, has been recruited to stabilise the blockage of the pyrimidine site); and (iv) the absence of any precise structural counterparts for two important purine-binding residues of RNase A. Analysis of the native structures has revealed details of the cell-binding region and nuclear localisation signal of Ang that are critical for angiogenicity. The cell-binding site differs dramatically from the corresponding regions of RNase A and two other homologues, eosinophil-derived neurotoxin and onconase, all of which lack angiogenic activity. Determination of the structures of the catalytically inactive variants K40Q and H13A has now allowed a rigorous assessment of the relationship between the ribonucleolytic and biological activities of Ang. No significant change outside the enzymatic active site was observed in K40Q, establishing that the loss of angiogenic activity for this derivative is directly attributable to disruption of the catalytic apparatus. The H13A structure shows some changes beyond the ribonucleolytic site, but sites involved in cell-binding and nuclear translocation are essentially unaffected by the amino acid replacement.
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PMID:Refined crystal structures of native human angiogenin and two active site variants: implications for the unique functional properties of an enzyme involved in neovascularisation during tumour growth. 991 22

Several nonmammalian members of the RNase A superfamily exhibit anticancer activity that appears to correlate with resistance to the cytosolic ribonuclease inhibitor (RI). We mutated two human ribonucleases-pancreatic RNase (hRNAse) and eosinophil-derived neurotoxin (EDN)-to incorporate cysteine residues at putative sites of close contact to RI, but distant from the catalytic sites. Coupling of Cys89 of RNase and Cys87 of EDN to proteins at these sites via a thioether bond produced enzymatically active conjugates that were resistant to RI. To elicit cellular targeting as well as to block RI binding, transferrin was conjugated to a mutant human RNase, rhRNase(Gly89)-->Cys) and a mutant EDN (Thr87-->Cys). The transferrin-rhRNase(Gly89-->Cys) thioether conjugate was 5000-fold more toxic to U251 cells than recombinant wild-type hRNase. In addition, transferrin-targeted EDN exhibited tumor cell toxicities similar to those of hRNase. Thus, we endowed two human RI-sensitive RNases with greater cytotoxicity by increasing their resistance to RI. This strategy has the potential to generate a novel set of recombinant human proteins useful for targeted therapy of cancer.
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PMID:Engineering receptor-mediated cytotoxicity into human ribonucleases by steric blockade of inhibitor interaction. 1033 82

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' --> 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1-2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.
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PMID:Potent inhibition of mammalian ribonucleases by 3', 5'-pyrophosphate-linked nucleotides. 1032 90


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