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

---

Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There have been some attempts to develop immunotoxins utilizing human RNase as a cytotoxic domain of antitumor agents. We have recently shown that only human RNase 3 (eosinophil cationic protein, ECP) among five human pancreatic-type RNases excels in binding to the cell surface and has a growth inhibition effect on several cancer cell lines, even though the RNase activity of RNase 3 is completely inhibited by the ubiquitously expressed cytosolic RNase inhibitor. This phenomenon may be explained by that RNase 3 is very stable against proteolytic degradation because RNase 3 internalized through endocytosis could have a longer life time in the cytosol, resulting in the accumulation of enough of it to exceed the concentration of RNase inhibitor, which allows the degradation of cytosolic RNA molecules. Thus, we compared the stabilities of human pancreatic-type RNases (RNases 1-5) and bovine RNase A by means of guanidium chloride-induced denaturation experiments based on the assumption of a two-state transition for unfolding. It was demonstrated that RNase 3 is extraordinarily stabler than either RNase A or the other human RNases (by more than 25 kJ/mol). Thus, our data suggest that in addition to its specific affinity for certain cancer cell lines, the stability of RNase 3 contributes to its unique cytotoxic effect and that it is important to stabilize a human RNase moiety through protein engineering for the design of human RNase-based immunotoxins.
...
PMID:RNase 3 (ECP) is an extraordinarily stable protein among human pancreatic-type RNases. 1241 23

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.
...
PMID:A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action. 1270 57

Attempts have been made to prepare rat liver microsomes and ribosomes free of RNase activity. Washing of microsomes with a large number of reagents, as well as preparation of microsomes by homogenizing the liver in the presence of a variety of reagents chosen to remove or inhibit RNase activity, failed to abolish completely the enzyme activity. However, when rat liver was homogenized in the presence of optimal concentrations of ATP the microsomes subsequently obtained showed no RNase activity. The composition of such microsomes was compared to controls prepared without the use of ATP. Preparation of microsomes with the use of ATP apparently repressed but did not remove the RNase activity for, when such microsomes were treated with 1 per cent deoxycholate to obtain ribosomes, the latter exhibited normal RNase activity. A possible explanation for these results based on several experiments is given. The incorporation of C(14) of L-leucine-C(14) into control and ATP-treated microsomes was measured. Repression of RNase activity by use of ATP or with RNase inhibitor, significantly reduced the incorporation. As a result of these and other experiments it is tentatively concluded that an alkaline RNase is a normal constituent of rat liver ribosomes and plays a role in the biological activity of these particles.
...
PMID:Studies on the function of intracellular ribonucleases. III. The relationship of the ribonuclease activity of rat liver microsomes to their biological activity. 1374 41

Basic fibroblast growth factor (bFGF) was inserted in the middle of human ribonuclease 1 (RNase1) sequence at an RNase inhibitor (RI)-binding site (Gly89) by a new gene fusion technique, insertional-fusion. The resultant insertional-fusion protein (CL-RFN89) was active both as bFGF and as RNase. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI-binding caused by fused bFGF domain. As a result, CL-RFN89 showed stronger growth inhibition on B16/BL6 melanoma cells than an RI-sensitive tandem fusion protein. Thus, the insertional-fusion technique increases accessible positions for gene fusion on RNase, resulting in construction of a potent cytotoxic RNase.
...
PMID:Insertional-fusion of basic fibroblast growth factor endowed ribonuclease 1 with enhanced cytotoxicity by steric blockade of inhibitor interaction. 1519 17

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.
...
PMID:Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity. 1527 33

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. The experiment demonstrated that it might effectively inhibit tumor-induced angiogenesis and inhibit tumor growth. Ribonuclease inhibitor is constructed almost entirely of leucine-rich repeats, which might be involved in unknown biological effects besides inhibiting RNase A and angiogenin activities. The exact molecular mechanism of antitumor on ribonuclease inhibitor remains unclear so far. In order to further understand the function of ribonuclease inhibitor and investigate the relationship with tumor growth, our study established a transfection of human ribonuclease inhibitor cDNA into the murine B16 cells by the retroviral packaging cell line PA317. The cell line transfected with a stably high expression of ribonuclease inhibitor was identified. We found that the transfected ribonuclease inhibitor could obviously inhibit cell proliferation, regulate cell cycle and induce cell apoptosis in vitro. Mice that were injected with the B16 cells transfected RI cDNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower density of microvessels, longer latent periods, and survival time than those in the other two control groups. In conclusion, the results reveal the novel mechanism that antitumor effect of ribonuclease inhibitor is also associated with inducing apoptosis, regulating cell cycle and inhibiting proliferation besides antiangiogenesis. These results suggest that ribonuclease inhibitor might be a candidate of tumor suppressor gene in some tissues. RI could become a target gene for gene therapy. Our study may be of biological and clinical importance.
...
PMID:Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells. 1577 86

Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence. In the present paper, two molecules of alanine to substitute for cys328/cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K, and then transformed Pichia pastoris GS115 by electroporation. After colony screening , the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.
...
PMID:[Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris]. 1584 55

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.
...
PMID:Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans. 1664 92

Human ribonuclease-1 (hRNase-1) is an extracellular enzyme found in exocrine pancreas, blood, milk, saliva, urine and seminal plasma, which has been implicated in digestion of dietary RNA and in antiviral host defense. The enzyme is characterized by a high catalytic activity toward both single-stranded and double-stranded RNA. In this study, we explored the possibility that hRNase-1 may also be provided with a ribonuclease H activity, i.e. be able to digest the RNA component of RNA:DNA hybrids. For this purpose, we developed an accurate and sensitive real-time RNase H assay based on a fluorogenic substrate made of a 12 nt 5'-fluorescein-labeled RNA hybridized to a complementary 3'-quencher-modified DNA. Under physiological-like conditions, hRNase-1 was found to cleave the RNA:DNA hybrid very efficiently, as expressed by a kcat/K(m) of 330 000 M(-1) s(-1), a value that is over 180-fold higher than that obtained with the homologous bovine RNase A and only 8-fold lower than that measured with Escherichia coli RNase H. The kinetic characterization of hRNase-1 showed that its hybridase activity is maximal at neutral pH, increases with lowering ionic strength and is fully inhibited by the cytosolic RNase inhibitor. Overall, the reported data widen our knowledge of the enzymatic properties of hRNase-1 and provide new elements for the comprehension of its biological function.
...
PMID:Hybridase activity of human ribonuclease-1 revealed by a real-time fluorometric assay. 1673 29

In this study, we explore the evolution and function of two closely related RNase A ribonucleases from the chicken, Gallus gallus. Separated by approximately 10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (dN > dS) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both approximately 16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (kcat = 2.6 and 0.056 s(-1), respectively), and surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI 11.0), is both angiogenic and bactericidal; RNase A-1 (pI 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71-76) and III (amino acids 89-104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, nonribonucleolytic proteins.
...
PMID:Evolution and function of leukocyte RNase A ribonucleases of the avian species, Gallus gallus. 1680 91


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---