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)

Domain swapping, the process in which a structural unit is exchanged between monomers to create a dimer containing two subunits of the monomeric fold, is believed to be an important mechanism for oligomerization and the formation of amyloid fibrils. Structural studies have implicated proline as an important residue for domain swapping due to its increased frequency in hinge regions preceding swapped arms. We hypothesized that proline's unique ability to populate both cis and trans peptide bond conformations may allow proline to act as a conformational gatekeeper, regulating interconversion between monomer and domain-swapped dimer forms. The hinge region of RNase A contains a proline at residue 114 that adopts a cis conformation in the monomer and extends to a trans conformation in the C-terminal domain-swapped dimer. Substitution of P114 with residues that strongly prefer a trans peptide bond (Ala, Gly) results in significant population of the C-terminal domain-swapped dimer under near-physiological conditions (pH 8.0, 37 degrees C). This is in stark contrast to dimerization of wild-type RNase A, which requires incubation under extreme conditions such as lyophilization from acetic acid or elevated temperature. In addition, we observe similar results when cis-P114 is mutated to glycine in a homologous RNase, human pancreatic RNase 1. Our results suggest that isomerization at P114 may facilitate population of a partially unfolded intermediate or alternative structure competent for domain swapping and provide support for a more general role for proline isomerization as a conformational gatekeeper in domain swapping and oligomerization.
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PMID:A hinge region cis-proline in ribonuclease A acts as a conformational gatekeeper for C-terminal domain swapping. 2047 98

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.
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PMID:RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage. 2067 93

Previously, VpPR-10.1 was isolated and characterized from a cDNA library of a fungus-resistant accession of Chinese wild grape (Vitis pseudoreticulata). We found that expression of VpPR-10.1 is affected by the fungal pathogen Erysiphe necator. To investigate the biochemical basis of the nuclease activity of VpPR-10.1 and its role in antifungal resistance, we generated recombinant VpPR-10.1 as well as site-directed mutations targeting three conserved amino acid residues among plant PR-10 s: Lys55, Glu149, and Tyr151. We showed that wild-type recombinant VpPR-10.1 exhibits both RNase and DNase activities. Mutant VpPR10.1-Y151H essentially retained all these activities. In contrast, VpPR10.1-K55N, where Lys55 in the P-loop region is mutated to Asn, and VpPR10.1-E149G, where Glu149 is mutated to Gly, lost their nuclease activity, indicating that both residues play a critical role in catalyzing RNA and DNA degradation. Furthermore, VpPR10.1 and VpPR10.1-Y151H inhibited the growth of the cultured fungal pathogen Alternaria alternate. Through transient expression in grapevine, we also demonstrated that VpPR10.1-K55N and VpPR10.1-E149G compromised resistance to E. necator. Finally, we further found that VpPR-10.1 can lead to programmed cell death and DNA degradation when incubated with tobacco BY-2 suspension cells. We show here that Lys55 and Glu149, but not Tyr151, are required for the RNase, DNase and antifungal activities of VpPR-10.1. The strong correlation between the level of VpPR-10.1 nuclease activity and its antifungal property indicates that the former is the biochemical basis for the latter. Taken together, our experiments revealed that VpPR-10.1 is critical in mediating fungal resistance in grape, potentially playing a dual role by degrading pathogen RNA and inducing programmed death of host cells.
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PMID:A pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: an insight of its mode of antifungal activity. 2475 5

Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean 'Suinong 10' infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homology of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved 'P-loop' (phosphate-binding loop) motif at residues 47-55 aa and a Bet v 1 domain at residues 87-120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible 'Dongnong 50' soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.
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PMID:A Novel Pathogenesis-Related Class 10 Protein Gly m 4l, Increases Resistance upon Phytophthora sojae Infection in Soybean (Glycine max [L.] Merr.). 2647 89

Commercial uses of bioactive peptides require low cost, effective methods for their production. We developed a new carrier protein for high yield production of recombinant peptides in Escherichia coli very well suited for the production of toxic peptides like antimicrobial peptides. GKY20, a short antimicrobial peptide derived from the C-terminus of human thrombin, was fused to the C-terminus of Onconase, a small ribonuclease (104 amino acids), which efficiently drove the peptide into inclusion bodies with very high expression levels (about 200-250 mg/L). After purification of the fusion protein by immobilized metal ion affinity chromatography, peptide was obtained by chemical cleavage in diluted acetic acid of an acid labile Asp-Pro sequence with more than 95% efficiency. To improve peptide purification, Onconase was mutated to eliminate all acid labile sequences thus reducing the release of unwanted peptides during the acid cleavage. Mutations were chosen to preserve the differential solubility of Onconase as function of pH, which allows its selective precipitation at neutral pH after the cleavage. The improved carrier allowed the production of 15-18 mg of recombinant peptide per liter of culture with 96-98% purity without the need of further chromatographic steps after the acid cleavage. The antimicrobial activity of the recombinant peptide, with an additional proline at the N-terminus, was tested on Gram-negative and Gram-positive strains and was found to be identical to that measured for synthetic GKY20. This finding suggests that N-terminal proline residue does not change the antimicrobial properties of recombinant (P)GKY20. The improved carrier, which does not contain cysteine and methionine residues, Asp-Pro and Asn-Gly sequences, is well suited for the production of peptides using any of the most popular chemical cleavage methods.
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PMID:Rational Design of a Carrier Protein for the Production of Recombinant Toxic Peptides in Escherichia coli. 2680 36

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.
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PMID:Unraveling the essential role of CysK in CDI toxin activation. 2753 61

Angiogenin (ANG) is a secretory ribonuclease that promotes the proliferation of endothelial cells, leading to angiogenesis. This function relies on its ribonucleolytic activity, which is low for simple RNA substrates. Upon entry into the cytosol, ANG is sequestered by the ribonuclease inhibitor protein (RNH1). We find that ANG is a potent cytotoxin for RNH1-knockout HeLa cells, belying its inefficiency as a nonspecific catalyst. The toxicity does, however, rely on the ribonucleolytic activity of ANG and a cytosolic localization, which lead to the accumulation of particular tRNA fragments (tRFs), such as tRF-5 Gly-GCC. These up-regulated tRFs are highly cytotoxic at physiological concentrations. Although ANG is well-known for its promotion of cell growth, our results reveal that ANG can also cause cell death.
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PMID:Human angiogenin is a potent cytotoxin in the absence of ribonuclease inhibitor. 2974 93

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3'-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH2-Gly(ArgLeu)4-TCAA displaying ribonuclease activity to its 5'-end. Designed miRNases efficiently cleaved miRNA targets, exhibiting Pyr-X specificity, and cleavage specificity had strong dependence on the miRNA sequence in the site of peptide location. In vitro, designed miRNases do not prevent cleavage of miRNA bound with the conjugate by RNase H, and more than an 11-fold enhancement of miRNA cleavage by the conjugate is observed in the presence of RNase H. In murine melanoma cells, miRNase silences mmu-miR-17 with very high efficiency as a result of miR-17 cleavage by miRNase and by recruited RNase H. Thus, miRNases provide a system of double attack of the miRNA molecules, significantly increasing the efficiency of miRNA downregulation in the cells in comparison with antisense oligonucleotide.
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PMID:Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H. 3030 12

tRNA fragments (tRFs) and tRNA halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more. Overexpressed angiogenin (ANG) cleaves tRNA anticodons and produces tRNA halves similar to those produced in response to stress. However, it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRFs are generated from the tRNA halves. Here, using global short RNA-Seq approach, we found that ANG overexpression selectively cleaves a subset of tRNAs, including tRNAGlu, tRNAGly, tRNALys, tRNAVal, tRNAHis, tRNAAsp, and tRNASeC to produce tRNA halves and tRF-5s that are 26-30 bases long. Surprisingly, ANG knockout revealed that the majority of stress-induced tRNA halves, except for the 5' half from tRNAHisGTG and the 3' half from tRNAAspGTC, are ANG independent, suggesting there are other RNases that produce tRNA halves. We also found that the 17-25 bases-long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from tRNA halves. Consistent with this, ANG knockout did not decrease tRF-3 levels or gene-silencing activity. We conclude that ANG cleaves specific tRNAs and is not the only RNase that creates tRNA halves and that the shorter tRFs are not generated from the tRNA halves or from independent tRNA cleavage by ANG.
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PMID:Angiogenin generates specific stress-induced tRNA halves and is not involved in tRF-3-mediated gene silencing. 3158 61

Agricultural production is becoming increasingly dependent on the environmental factors that alter soil properties, plant productivity, and product quality. Environment pollution caused by heavy metals because of human activities are among the most dangerous pollutants on the biosphere. Here, we have studied the biochemical adaptation of wild and cultivated soybeans to the simulated effects of lead nitrate and lead acetate. Lead in the form of acetate had a relevant toxic effect, as evidenced by a significant increase in the concentration of malonic dialdehyde in the treated samples relative to control samples. Catalase and peroxidase, possibly performing a signaling function, are involved in the adaptation to the toxicity of Pb salts. The studied Pb salts showed a predominant stimulating effect on the specific activity of acid phosphatases in cultivated soybean, while the ribonuclease activity changed in both Glycine species. Moreover, in wild soybean, it was mostly suppressive, except for the first day. We found that the electrophoretic spectra of acid phosphatases of soybean seedlings was highly stabile, while that of ribonucleases varied depending on the salt. On the seventh day of exposure, lead nitrate caused a decrease in the specific activity of the studied hydrolases of seedlings of cultivated and wild soybeans. A change in the number or electrophoretic mobility of multiple forms of enzymes during treatment with Pb salts was revealed, which indicates the adaptation of the plants at the molecular genetic level. These results imply that the observed enzymes can be used as sensitive indicators for predicting the effects of heavy metals on soybean.
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PMID:Biochemical adaptation of wild and cultivated soybean against toxicity of lead salts. 3247 23


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