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 pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.
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PMID:Dimerization of glycoprotein E(rns) of classical swine fever virus is not essential for viral replication and infection. 1598 75

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.
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PMID:Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily. 1619 69

1. The reaction between beta-bromopyruvic acid and SH groups of cysteine residues in reduced ribonuclease and in some other polypeptides was investigated. 2. One molecule of the acid was found to be necessary to block one SH group in reduced ribonuclease. The stoicheiometry of the interaction and the spectral characteristics of the compound formed suggested that the product is and S-oxalomethyl (R.S.CH(2).CO.CO(2)H) derivative of reduced ribonuclease. 3. Digestion of reduced S-oxalomethylated ribonuclease by trypsin or chymotrypsin induced changes in the spectrum that could be attributed to the liberation of the alpha-amino group of S-oxalomethylated cysteine residues from peptide bonds. The spectral changes that accompanied the hydrolysis of specific peptide bonds in reduced S-oxalomethylated ribonuclease and S-oxalomethylated co-poly(l-Lys,l-CySH) allowed the kinetics of the digestion to be followed. 4. Possible applications of the spectrophotometric method in the study of protein structure are discussed.
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PMID:Spectrophotometric measurement of the liberation of the alpha-amino group of cysteine residues in polypeptides. 1674 6

The family of small leucine-rich repeat proteins and proteoglycans (SLRPs) contains several extracellular matrix molecules that are structurally related by a protein core composed of leucine-rich repeats (LRRs) flanked by two conserved cysteine-rich regions. The small proteoglycan decorin is the archetypal SLRP. Decorin is present in a variety of connective tissues, typically "decorating" collagen fibrils, and is involved in important biological functions, including the regulation of the assembly of fibrillar collagens and modulation of cell adhesion. Several SLRPs are known to regulate collagen fibrillogenesis and there is evidence that they may share other biological functions. We have recently determined the crystal structure of the protein core of decorin, the first such determination of a member of the SLRP family. This structure has highlighted several correlations: (1) SLRPs have similar internal repeat structures; (2) SLRP molecules are far less curved than an early model of decorin based on the three-dimensional structure of ribonuclease inhibitor; (3) the N-terminal and C-terminal cysteine-rich regions are conserved capping motifs. Furthermore, the structure shows that decorin dimerizes through the concave surface of its LRR domain, which has been implicated previously in its interaction with collagen. We have established that both decorin and opticin, another SLRP, form stable dimers in solution. Conservation of residues involved in decorin dimerization suggests that the mode of dimerization for other SLRPs will be similar. Taken together these results suggest the need for reevaluation of currently accepted models of SLRP interaction with their ligands.
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PMID:Structural correlations in the family of small leucine-rich repeat proteins and proteoglycans. 1688 25

The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local electric field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: ribonuclease S (RNase S), human aldose reductase (hALR2), and the reaction center (RC) of Rhodobacter capsulatus, through a facile synthetic scheme for the transformation of cysteine residues into thiocyanatoalanine. Vibrational Stark effect spectroscopy and Fourier transform infrared spectroscopy on the modified proteins demonstrated that thiocyanate residues are a highly general tool for probing electrostatic fields in proteins.
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PMID:Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins. 1703 38

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.
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PMID:Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes. 1756 Jun 6

The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.
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PMID:Crystal structure of type 1 ribonuclease H from hyperthermophilic archaeon Sulfolobus tokodaii: role of arginine 118 and C-terminal anchoring. 1789 5

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of coevolution higher than that for nonobligate protein-protein interactions. The coevolution of the proteins involved in certain nonobligate interactions is, however, essential to cell survival. To gain insight into the coevolution of one such nonobligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.
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PMID:Intraspecies regulation of ribonucleolytic activity. 1795 29

Glutathione depletion is a key factor in the development of acute pancreatitis. Our aim was to study the regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in edematous or necrotizing pancreatitis in rats. Glutathione levels were kept low in necrotizing pancreatitis for several hours, with no increase in protein or mRNA levels of glutamate cysteine ligase subunits, despite binding of RNA polymerase II to their promoters and coding regions. The survival signal pathway mediated by ERK and c-MYC was activated, and c-MYC was recruited to the promoters. The failure in gene up-regulation seems to be due to a marked increase in cytosolic ribonuclease activity. In contrast, in edematous pancreatitis glutathione levels were depleted and rapidly restored, and protein and mRNA expression of glutamate cysteine ligase increased markedly due to enhanced transcription mediated by recruitment of c-MYC, NF-kappaB, and SP-1 to the promoters. No increase in cytosolic ribonuclease activity was found in this case. We propose a novel pathophysiological mechanism to differentiate necrotizing from edematous pancreatitis, which is the inefficient up-regulation of glutamate cysteine ligase caused by increased cytosolic ribonuclease activity in the severe form of the disease. This mechanism would abrogate a rapid recovery of glutathione levels.
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PMID:Glutamate cysteine ligase up-regulation fails in necrotizing pancreatitis. 1827 77

Bovine pancreatic deoxyribonuclease I (bpDNase I) contains four cysteine residues forming two disulfide bonds. Though there are no free sulfhydryl groups, incubation of bpDNase I with 2-nitro-5-thiosulfobenzoic acid (NTSB) in the presence of Ca(2+) or Mg(2+) at pH 7.5 results in inactivation of the enzyme. Amino acid analysis shows that NTSB-treated bpDNase I still contains all 4 half-cystine residues. The only amino acid residues having reduced values are threonine and serine, indicating that these may be the reaction sites for NTSB. Plasmid scission assay and circular dichroism analysis reveal the structural integrity of the inactivated enzyme. Treatment of bpDNase I with NTSB does not result in fragmentation, as demonstrated by SDS-PAGE analysis. NTSB binds bpDNase I through covalent modification, since dialysis and gel filtration can not reverse the inactivation reaction. However, after dilution into an acid buffer of pH 4.7, the inactivated enzyme regains about 40% of its initial activity, suggesting a reversible inactivation by acid treatment. NTSB does not inactivate DNase II, ribonuclease, chymotrypsin and lysozyme, while it effectively inactivates rat parotid DNase I. These results strongly suggest that NTSB can be considered as a novel inhibitor specific for DNase I.
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PMID:2-nitro-5-thiosulfobenzoic acid as a novel inhibitor specific for deoxyribonuclease I. 1829 70


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