The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Ronald T. Raines - One of the best experts on this subject based on the ideXlab platform.
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knockout of the ribonuclease inhibitor gene leaves human cells vulnerable to secretory Ribonucleases
Biochemistry, 2016Co-Authors: Sydney P Thomas, Ronald T. RainesAbstract:Ribonuclease inhibitor (RNH1) is a cytosolic protein that binds with femtomolar affinity to human ribonuclease 1 (RNase 1) and homologous secretory Ribonucleases. RNH1 contains 32 cysteine residues and has been implicated as an antioxidant. Here, we use CRISPR-Cas9 to knock out RNH1 in HeLa cells. We find that cellular RNH1 affords marked protection from the lethal ribonucleolytic activity of RNase 1 but not from oxidants. We conclude that RNH1 protects cytosolic RNA from invading Ribonucleases.
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Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.
The Journal of biological chemistry, 2014Co-Authors: Chelcie H. Eller, Jo E. Lomax, Ronald T. RainesAbstract:Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among Ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.
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Contribution of electrostatics to the binding of pancreatic-type Ribonucleases to membranes.
Biochemistry, 2013Co-Authors: Nadia K. Sundlass, Chelcie H. Eller, Qiang Cui, Ronald T. RainesAbstract:Pancreatic-type Ribonucleases show clinical promise as chemotherapeutic agents but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of Ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of Ribonucleases. We focus on three homologous Ribonucleases: Onconase (frog), ribonuclease A (cow), and ribonuclease 1 (human). Our results support the hypothesis that electrostatics are necessary for the cellular uptake of Onconase. In contrast, specific interactions with cell-surface components likely contribute more to the cellular uptake of ribonuclease A and ribonuclease 1 than do electrostatics. These findings provide insight for the design of new cytotoxic Ribonucleases.
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variants of ribonuclease inhibitor that resist oxidation
Protein Science, 2008Co-Authors: Wayne L Schultz, Ronald T. RainesAbstract:Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type Ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328–Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type Ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
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evasion of ribonuclease inhibitor as a determinant of ribonuclease cytotoxicity
Current Pharmaceutical Biotechnology, 2008Co-Authors: Thomas J Rutkoski, Ronald T. RainesAbstract:Onconase® (ONC) is an amphibian member of the bovine pancreatic ribonuclease (RNase A) superfamily that exhibits innate antitumoral activity. ONC has been granted both orphan-drug and fast-track status by the U.S. Food and Drug Administration for the treatment of malignant mesothelioma, and is poised to become the first chemotherapeutic agent based on a ribonuclease. Investigations into the mechanism of ribonuclease-based cytotoxicity have elucidated several important determinants for cytotoxicity, including efficient deliverance of ribonucleolytic activity to the cytosol and preservation of conformation stability. Nevertheless, the most-striking similarity between ONC and bovine seminal ribonuclease, another naturally cytotoxic ribonuclease, is their insensitivity to inhibition by the potent cytosolic ribonuclease inhibitor protein (RI). RI typically binds to its ribonuclease ligands with femtomolar affinity—an extraordinary feat considering the lack of sequence identity among the bound Ribonucleases. Mammalian Ribonucleases such as RNase A or its human homologue, RNase 1, have the potential to be more desirable chemotherapeutic agents than ONC owing to their higher catalytic activity, low potential for immunogenicity, favorable tissue distribution, and high therapeutic index, but are limited by their sensitivity to RI. These non-toxic mammalian Ribonucleases can be transformed into potent cytotoxins by engendering them with RI-evasion using protein engineering strategies such as site-directed mutagenesis, multimerization, fusion to a targeting moiety, and chemical modification. In several instances, these engineered Ribonucleases exhibit greater cytotoxicity in vitro than does ONC. Herein, we review the biochemical characteristics of RI·ribonuclease complexes and progress towards the development of mammalian ribonuclease-based chemotherapeutics through the elicitation of RI-evasion.
Bert L Vallee - One of the best experts on this subject based on the ideXlab platform.
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An Angiogenic Protein from Bovine Serum and Milk — Purification and Primary Structure of Angiogenin-2
FEBS Journal, 1997Co-Authors: Daniel J. Strydom, Michael D Bond, Bert L ValleeAbstract:Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross-linked by three disulfide bonds, is glycosylated at Asn33, and is 57% identical to bovine angiogenin. The amino-terminal and carboxyl terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub-family of pancreatic Ribonucleases, and is referred to as angiogenin-2.
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an angiogenic protein from bovine serum and milk purification and primary structure of angiogenin 2
FEBS Journal, 1997Co-Authors: Daniel J. Strydom, Michael D Bond, Bert L ValleeAbstract:Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross-linked by three disulfide bonds, is glycosylated at Asn33, and is 57% identical to bovine angiogenin. The amino-terminal and carboxyl terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub-family of pancreatic Ribonucleases, and is referred to as angiogenin-2.
Johann Deisenhofer - One of the best experts on this subject based on the ideXlab platform.
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mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease a
Journal of Molecular Biology, 1996Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse Ribonucleases. However, the inhibition is lost with amphibian Ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited
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crystal structure of porcine ribonuclease inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:RIBONUCLEASE inhibitor is a cytoplasmic protein that tightly binds and inhibits Ribonucleases of the pancreatic ribonuclease super-family1. The primary sequence of this inhibitor contains leucine-rich repeats2 (LRRs); these motifs are present in many proteins that participate in protein–protein interactions and have different functions and cellular locations. In vivo, ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells3 and in angiogenesis4. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of ribonuclease inhibitor with Ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of α/β protein fold. Individual repeats constitute β–α structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel β-sheet may explain why LRRs are used to achieve strong protein–protein interactions. A possible ribonuclease-binding region incorporates the surface formed by the parallel β-sheet and the βα loops.
Bestjan Kobe - One of the best experts on this subject based on the ideXlab platform.
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mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease a
Journal of Molecular Biology, 1996Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse Ribonucleases. However, the inhibition is lost with amphibian Ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited
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crystal structure of porcine ribonuclease inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:RIBONUCLEASE inhibitor is a cytoplasmic protein that tightly binds and inhibits Ribonucleases of the pancreatic ribonuclease super-family1. The primary sequence of this inhibitor contains leucine-rich repeats2 (LRRs); these motifs are present in many proteins that participate in protein–protein interactions and have different functions and cellular locations. In vivo, ribonuclease inhibitor may have a role in the regulation of RNA turnover in mammalian cells3 and in angiogenesis4. To define the structural features of LRR proteins and to understand better the nature of the tight interaction of ribonuclease inhibitor with Ribonucleases, we have determined the crystal structure of the porcine inhibitor. To our knowledge, this is the first three-dimensional structure of a protein containing LRRs and represents a new class of α/β protein fold. Individual repeats constitute β–α structural units that probably also occur in other proteins containing LRRs. The non-globular shape of the structure and the exposed face of the parallel β-sheet may explain why LRRs are used to achieve strong protein–protein interactions. A possible ribonuclease-binding region incorporates the surface formed by the parallel β-sheet and the βα loops.
Jaap J. Beintema - One of the best experts on this subject based on the ideXlab platform.
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The primary structure of muskrat pancreatic ribonuclease.
International journal of peptide and protein research, 2009Co-Authors: H. Van Dijk, B. Sloots, A.m. Van Den Berg, Wim Gaastra, Jaap J. BeintemaAbstract:Pancreatic ribonuclease from muskrat (Ondatra zibethica) was isolated and its amino acid sequence was determined from tryptic digests of the performic acid-oxidized and the reduced and aminoethylated enzyme. The peptides have been positioned in the sequence by homology with other Ribonucleases. This could be done unambiguously for all peptides except Arg-Arg (tentative position 32–33) and Ser-Arg (tentative position 75–76). The amino acid sequences of the peptides were determined by the dansyl-Edman method, with the exception of residues 23–25 and 99–102, which were positioned by homology. The enzyme differs in 38 positions from the enzyme from rat and in 31–42 positions from other mammalian pancreatic Ribonucleases, while rat ribonuclease differs at 44–52 positions from the other enzymes. These data point to a common ancestry of the enzymes from muskrat and rat and an increased evolution rate of rat ribonuclease after divergence of the ancestors of both species. Muskrat ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn- Val-Thr (62–64).
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Inclusion of Cetaceans within the order Artiodactyla based on phylogenetic analysis of pancreatic ribonuclease genes
Journal of Molecular Evolution, 1999Co-Authors: Reinhard G. Kleineidam, Heleen J. Breukelman, Graziano Pesole, Jaap J. Beintema, Robert A. KasteleinAbstract:Mammalian secretory Ribonucleases (RNases 1) form a family of extensively studied homologous proteins that were already used for phylogenetic analyses at the protein sequence level previously. In this paper we report the determination of six ribonuclease gene sequences of Artiodactyla and two of Cetacea. These sequences have been used with ruminant homologues in phylogenetic analyses that supported a group including hippopotamus and toothed whales, a group of ruminant pancreatic and brain-type Ribonucleases, and a group of tylopod sequences containing the Arabian camel pancreatic ribonuclease gene and Arabian and Bactrian camel and alpaca RNase 1 genes of unknown function. In all analyses the pig was the first diverging artiodactyl. This DNA-based tree is compatible to published trees derived from a number of other genes. The differences to those trees obtained with ribonuclease protein sequences can be explained by the influence of convergence of pancreatic RNases from hippopotamus, camel, and ruminants and by taking into account the information from third codon positions in the DNA-based analyses. The evolution of sequence features of Ribonucleases such as the distribution of positively charged amino acids and of potential glycosylation sites is described with regard to increased double-stranded RNA cleavage that is observed in several cetacean and artiodactyl RNases which may have no role in ruminant or ruminant-like digestion.
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Introduction: the ribonuclease A superfamily
Cellular and molecular life sciences : CMLS, 1998Co-Authors: Jaap J. BeintemaAbstract:In this multi-author issue several aspects of the ribonuclease A superfamily are reviewed. This superfamily can be subdivided in a number of mammalian and other vertebrate ribonuclease families. In the introduction chapter the titles of the other contributions are presented. There is little uniformity in the nomenclature of Ribonucleases, caused in part by gene duplications, which have occurred independently in several mammalian lineages, and which are nice examples for explaining orthology and paralogy in molecular evolution.
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The ribonuclease A superfamily : general discussion
Cellular and molecular life sciences : CMLS, 1998Co-Authors: Jaap J. Beintema, Reinhard G. KleineidamAbstract:Enzymic properties of members of the ribonuclease A superfamily, like the activity on RNA, the preference for either cytosine or uracil in the primary binding site B1 , the preference for the other side of the cleaved phosphodiester bond, the B2 site, and features of the two noncatalytic phosphate-binding sites P0 and P2 are discussed in several articles in this multi-author review, and are summarized in this closing article. A special feature of members of the Ribonucleases 1 family is their destabilizing action on double-stranded nucleic acid structures. A feature of the ribonuclease A superfamily is the frequent occurrence of gene duplications, both in ancestral vertebrate lineages and in recently evolved taxa. Three different bovine Ribonucleases 1 have been identified in pancreas, semen and brain, respectively, which are the result of two gene duplications in an ancestral ruminant. Similar gene duplications have been identified in other ribonuclease families in several mammalian and other vertebrate taxa. The ribonuclease family, of which the human members have been assigned numbers 2, 3 and 6, underwent a still mysterious pattern of gene duplications and functional expression as proteins with ribonuclease activity and other physiological properties.
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high sequence similarity between a ribonuclease from ginseng calluses and fungus elicited proteins from parsley indicates that intracellular pathogenesis related proteins are Ribonucleases
Planta, 1994Co-Authors: Gennady P Moiseyev, Jaap J. Beintema, Larisa I Fedoreyeva, Gennady I YakovlevAbstract:A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other Ribonucleases was found, but there was 60–70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are Ribonucleases.
