Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.
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PMID:Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis. 169 38

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin. 169 54

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.
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PMID:A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis. 271 39

Human angiogenin (Ang), a homologue of bovine pancreatic ribonuclease A (RNase A), is a potent inducer of blood vessel formation. It exerts a ribonucleolytic activity that is 10(5)-10(6)-fold lower than that of RNase A but nonetheless essential for biological action. Previous studies revealed some of the structural features of Ang that underlie its catalytic inefficiency: Gln-117 blocks the space corresponding to the pyrimidine binding site of RNase A and Ang lacks the disulfide loop 65-72 that forms most of the purine binding site of RNase A. Additional features have now been identified by mutagenesis and kinetics. Thr-80, which hydrogen-bonds to the pyrimidine-binding residue Thr-44, plays an important part in attenuating activity and in determining pyrimidine specificity: mutation to Ala increases activity toward cytidylyl substrates by 11-15-fold but has only a minimal effect on cleavage of uridylyl substrates. The properties of T44A/T80A and Q117A/T80A double mutants demonstrate that these changes are mediated by Thr-44 and are largely independent of the blockage by Gln-117. The side chain of Ser-118 also suppresses enzymatic activity: S118A is 5-7-fold more effective than Ang. This increase appears to reflect the loss of a hydrogen bond with Asp-116 that helps to orient Gln-117. The effects of deleting residues 119-123 suggest that main-chain atoms of the C-terminal 3(10) helix make a small further contribution. Finally, the significance of the absence of the RNase A loop 65-72 from Ang has been investigated by reexamining the earlier derivative ARH-I (in which Ang residues 58-70 have been replaced by residues 59-73 of RNase) and generating new derivatives of this hybrid protein. The results suggest that the RNase A segment of ARH-I not only provides more effective purine recognition but also counteracts the deleterious effects of Gln-117 and Thr-80 on the pyrimidine site.
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PMID:Structural features that determine the enzymatic potency and specificity of human angiogenin: threonine-80 and residues 58-70 and 116-123. 957 71

Angiogenin and ribonuclease A share 33% sequence identity but have distinct functions. Angiogenin is a potent inducer of angiogenesis that is only weakly ribonucleolytic, whereas ribonuclease A is a robust ribonuclease that is not angiogenic. A chimera ("ARH-I"), in which angiogenin residues 58-70 are replaced with residues 59-73 of ribonuclease A, has intermediate ribonucleolytic potency and no angiogenic activity. Here we report a crystal structure of ARH-I that reveals the molecular basis for these characteristics. The ribonuclease A-derived (guest) segment adopts a structure largely similar to that in ribonuclease A, and successfully converts this region from a cell-binding site to a purine-binding site. At the same time, its presence causes complex changes in the angiogenin-derived (host) portion that account for much of the increased ribonuclease activity of ARH-I. Guest-host interactions of this type probably occur more generally in protein chimeras, emphasizing the importance of direct structural information for understanding the functional behavior of such molecules.
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PMID:Guest-host crosstalk in an angiogenin-RNase A chimeric protein. 1217 35