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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human angiogenin is a potent cytotoxin in the absence of Ribonuclease Inhibitor
RNA, 2018Co-Authors: Sydney P Thomas, Trish T Hoang, Valerie T Ressler, Ronald T RainesAbstract: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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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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functional evolution of Ribonuclease Inhibitor insights from birds and reptiles
Journal of Molecular Biology, 2014Co-Authors: Jo E Lomax, Christopher M Bianchetti, A Chang, G N Phillips, Ronald T RainesAbstract:Abstract Ribonuclease Inhibitor (RI) is a conserved protein of the mammalian cytosol. RI binds with high affinity to diverse secretory Ribonucleases (RNases) and inhibits their enzymatic activity. Although secretory RNases are found in all vertebrates, the existence of a non-mammalian RI has been uncertain. Here, we report on the identification and characterization of RI homologs from chicken and anole lizard. These proteins bind to RNases from multiple species but exhibit much greater affinity for their cognate RNases than for mammalian RNases. To reveal the basis for this differential affinity, we determined the crystal structure of mouse, bovine, and chicken RI·RNase complexes to a resolution of 2.20, 2.21, and 1.92 A, respectively. A combination of structural, computational, and bioinformatic analyses enabled the identification of two residues that appear to contribute to the differential affinity for RNases. We also found marked differences in oxidative instability between mammalian and non-mammalian RIs, indicating evolution toward greater oxygen sensitivity in RIs from mammalian species. Taken together, our results illuminate the structural and functional evolution of RI, along with its dynamic role in vertebrate biology.
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Ribonuclease Inhibitor regulates neovascularization by human angiogenin
Biochemistry, 2009Co-Authors: Kimberly A Dickson, Dongku Kang, Young Sam Kwon, Peter A Leland, Sooik Chang, Ronald T RainesAbstract:Human angiogenin (ANG) is a homologue of bovine pancreatic Ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity. To stimulate blood vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic Ribonuclease Inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 106-fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.
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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.
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: B 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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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: B 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 Å resolution. Ribonuclease A binds to the concave region of the Inhibitor protein comprising its parallel β-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 p1 phosphate-binding pocket of Ribonuclease A, where a sulfate ion remains bound. The 2550 Å2of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K(i) = 5.9 x 10-14M). 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.
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complex between bovine Ribonuclease a and porcine Ribonuclease Inhibitor crystallizes in a similar unit cell as free Ribonuclease Inhibitor
Journal of Molecular Biology, 1994Co-Authors: Bestjan Kobe, Johann DeisenhoferAbstract:We obtained three different morphologies of crystals of bovine Ribonuclease A and porcine Ribonuclease Inhibitor. X-ray quality crystals were grown in 1·3 M ammonium sulfate, 100 mM sodium acetate (pH 5·0) and 20 mM dithiothreitol at 21°C. These crystals have the symmetry of the tetragonal space group I4 with a=133·3 A and c =86·7 A and diffract to 2·5 A resolution; they have the same symmetry and only slightly different cell parameters than the crystals of free Ribonuclease Inhibitor. Polyacrylamide gel electrophoresis and the crystal density indicate that both Ribonuclease Inhibitor and Ribonuclease A are present in the crystals. Although small, crystals are suitable for three-dimensional structural analysis.
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crystal structure of porcine Ribonuclease Inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: B 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.
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crystallization and preliminary x ray analysis of porcine Ribonuclease Inhibitor a protein with leucine rich repeats
Journal of Molecular Biology, 1993Co-Authors: B Kobe, Johann DeisenhoferAbstract:Ribonuclease Inhibitor was purified from pig liver and crystallized as 21°C from solutions containing dithiothreitol as an additive and ammonium sulfate, lithium sulfate or combinations of both as precipitants. Crystals have the symmetry of the tetragonal space group I4 with a = 134·9 A and c = 83·6 A, and diffract to better than 3 A resolution. Self rotation functions and packing density of the crystals are consistent with two molecules in the asymmetric unit.
B 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: B 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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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: B 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 Å resolution. Ribonuclease A binds to the concave region of the Inhibitor protein comprising its parallel β-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 p1 phosphate-binding pocket of Ribonuclease A, where a sulfate ion remains bound. The 2550 Å2of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K(i) = 5.9 x 10-14M). 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.
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crystal structure of porcine Ribonuclease Inhibitor a protein with leucine rich repeats
Nature, 1993Co-Authors: B 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.
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crystallization and preliminary x ray analysis of porcine Ribonuclease Inhibitor a protein with leucine rich repeats
Journal of Molecular Biology, 1993Co-Authors: B Kobe, Johann DeisenhoferAbstract:Ribonuclease Inhibitor was purified from pig liver and crystallized as 21°C from solutions containing dithiothreitol as an additive and ammonium sulfate, lithium sulfate or combinations of both as precipitants. Crystals have the symmetry of the tetragonal space group I4 with a = 134·9 A and c = 83·6 A, and diffract to better than 3 A resolution. Self rotation functions and packing density of the crystals are consistent with two molecules in the asymmetric unit.
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Crystallization and preliminary X-ray analysis of porcine Ribonuclease Inhibitor, a protein with leucine-rich repeats
Journal of Molecular Biology, 1993Co-Authors: B Kobe, Johann DeisenhoferAbstract:Ribonuclease Inhibitor was purified from pig liver and crystallized at 21°C from solutions containing dithiothreitol as an additive and ammonium sulfate, lithium sulfate or combinations of both as precipitants. Crystals have the symmetry of the tetragonal space group I4 with a = 134.9 Å and c = 83.6 Å, and diffract to better than 3 Å resolution. Self rotation functions and packing density of the crystals are consistent with two molecules in the asymmetric unit. © 1993 Academic Press, Inc.
Jan Hofsteenge - One of the best experts on this subject based on the ideXlab platform.
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interaction of semisynthetic variants of rnase a with Ribonuclease Inhibitor
Protein Science, 2008Co-Authors: Ulf Neumann, Jan HofsteengeAbstract:Derivatives of Ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of Ribonuclease Inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.
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high level soluble production and characterization of porcine Ribonuclease Inhibitor
Protein Expression and Purification, 2001Co-Authors: Tony A Klink, Jan Hofsteenge, Anna M Vicentini, Ronald T RainesAbstract:Abstract Ribonucleases can be cytotoxic if they retain their ribonucleolytic activity in the cytosol. The cytosolic ribonucleolytic activity of Ribonuclease A (RNase A) and other pancreatic-type Ribonucleases is limited by the presence of excess Ribonuclease Inhibitor (RI). RI is a 50-kDa cytosolic scavenger of pancreatic-type Ribonucleases that competitively inhibits their ribonucleolytic activity. RI had been overproduced as inclusion bodies, but its folding in vitro is inefficient. Here, porcine RI (pRI) was overproduced in Escherichia coli using the trp promoter and minimal medium. This expression system maintains pRI in the soluble fraction of the cytosol. pRI was purified by affinity chromatography using immobilized RNase A and by anion-exchange chromatography. The resulting yield of 15 mg of purified RI per liter of culture represents a 60-fold increase relative to previously reported recombinant DNA systems. Differential scanning calorimetry was used to study the thermal denaturation of pRI, RNase A, and the pRI-RNase A complex. The conformational stability of the complex is greater than that of the individual components.
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19 – Ribonuclease Inhibitor
Ribonucleases, 1997Co-Authors: Jan HofsteengeAbstract:Publisher Summary This chapter provides an overview of Ribonuclease Inhibitor (RI). RI is an acidic 50-kDa protein, characterized by a high content of leucine and cysteine residues. It is not structurally related to the recently identified Inhibitor of 2-5A-dependent RNase, nor to the Inhibitors of microbial RNases. The presence of RI in biological samples has generally been determined using two types of assays. The observation that RI, or at least its activity, occurs in a wide range of animals and in every tissue or cell examined suggests an essential role for this protein. The gene encoding human RI (RNH) has been localized to chromosome 11p15.5. Long-range restriction mapping placed it close to the Harvey ras protooncogene (HRAS1). This chapter discusses biological properties, species and tissue distribution, and biological function of RI. The chapter also elaborates molecular biology of RI. Molecular properties of RI are also discussed. The chapter concludes with a discussion on applications of RI in protection of RNA, RNasc assay, and inhibition of angiogenesis.
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19 Ribonuclease Inhibitor
Ribonucleases#R##N#Structures and Functions, 1997Co-Authors: Jan HofsteengeAbstract:Publisher Summary This chapter provides an overview of Ribonuclease Inhibitor (RI). RI is an acidic 50-kDa protein, characterized by a high content of leucine and cysteine residues. It is not structurally related to the recently identified Inhibitor of 2-5A-dependent RNase, nor to the Inhibitors of microbial RNases. The presence of RI in biological samples has generally been determined using two types of assays. The observation that RI, or at least its activity, occurs in a wide range of animals and in every tissue or cell examined suggests an essential role for this protein. The gene encoding human RI (RNH) has been localized to chromosome 11p15.5. Long-range restriction mapping placed it close to the Harvey ras protooncogene (HRAS1). This chapter discusses biological properties, species and tissue distribution, and biological function of RI. The chapter also elaborates molecular biology of RI. Molecular properties of RI are also discussed. The chapter concludes with a discussion on applications of RI in protection of RNA, RNasc assay, and inhibition of angiogenesis.
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oxidation of sulfhydryl groups of Ribonuclease Inhibitor in epithelial cells is sufficient for its intracellular degradation
Journal of Biological Chemistry, 1996Co-Authors: Montserrat Blazquez, Jesus Fominaya, Jan HofsteengeAbstract:Abstract Ribonuclease Inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine Inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.
Bert L Vallee - One of the best experts on this subject based on the ideXlab platform.
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interaction of human placental Ribonuclease with placental Ribonuclease Inhibitor
Biochemistry, 1991Co-Authors: Robert Shapiro, Bert L ValleeAbstract:The interactions of human placental Ribonuclease Inhibitor (PRI) with bovine pancreatic Ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.
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the placental Ribonuclease Inhibitor rnh gene is located on chromosome subband 11p15 5
Genomics, 1990Co-Authors: Stanislawa Weremowicz, Cynthia C Morton, Bert L ValleeAbstract:Abstract The Ribonuclease Inhibitor from human placenta is a tight-binding Inhibitor of alkaline and neutral Ribonucleases, including the blood vessel-inducing protein, angiogenin. The location of the Inhibitor gene within the human genome has now been determined. Utilizing human-rodent hybrid cell lines, it was found on chromosome 11. The localization was refined to chromosome band 11p15 by in situ hybridization of the Ribonuclease Inhibitor cDNA to normal metaphase chromosomes. A further refinement was obtained by in situ hybridization of the probe to metaphase chromosomes from RPMI 8402 cells, a line containing a well-characterized translocation t(11;14)(p15;q11) with a chromosome 11 breakpoint between the insulin-like growth factor 2 (IGF2) and Harvey rat sarcoma viral oncogene homolog genes. This analysis has localized the Ribonuclease Inhibitor gene to chromosome subband 11p15.5, distal to the IGF2 gene.
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modular mutagenesis of human placental Ribonuclease Inhibitor a protein with leucine rich repeats
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Bert L ValleeAbstract:Abstract Human placental Ribonuclease Inhibitor (PRI) is a potent protein Inhibitor of pancreatic Ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit Ribonuclease A. Therefore, the angiogenin/Ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.
