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Harold A. Scheraga - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleases as models for understanding protein folding
2011Co-Authors: Harold A. ScheragaAbstract:This is a summary of the background, and some of the experimental research in my laboratory, on the physical chemical properties of proteins. The experimental studies were carried out to obtain information about the three-dimensional structure and folding/unfolding pathways of bovine Pancreatic Ribonuclease A and three of its structural homologs, Ribonuclease B, frog onconase, and bovine angiogenin.
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solution nmr evidence for a cis tyr ala peptide group in the structure of pro93ala bovine Pancreatic Ribonuclease a
Protein Science, 2008Co-Authors: Ying Xiong, William J. Wedemeyer, Harold A. Scheraga, Darmawi Juminaga, G V T Swapna, Gaetano T MontelioneAbstract:Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine Pancreatic Ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.
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the oxidative folding rate of bovine Pancreatic Ribonuclease is enhanced by a covalently attached oligosaccharide
Biochemistry, 2005Co-Authors: Mahesh Narayan, Harold A. ScheragaAbstract:Bovine Pancreatic Ribonuclease B (RNase B) differs from RNase A by the presence of an oligosaccharide moiety covalently attached to Asn 34. Oxidative folding studies of RNase B were carried out at different temperatures using DTTox as the oxidizing agent, and the results were compared with those for RNase A. The oxidative folding rates of RNase B are between 1.7 and 1.3 times faster than those of RNase A at the temperatures that were investigated. The folding pathways of RNase B were determined to be similar to those of RNase A in that two structured intermediates, each lacking one native disulfide bond, were found to populate the regeneration pathways at 25 °C and pH 8.3. The thermodynamic stabilities of these two glycosylated intermediates, and their rates of formation from their unstructured precursors in the rate-determining step, were found to be higher than those of their unglycosylated counterparts from RNase A. Thus, the underlying cause for the faster rate of oxidative regeneration of native RNas...
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interactions that favor the native over the non native disulfide bond among residues 58 72 in the oxidative folding of bovine Pancreatic Ribonuclease a
Biochemistry, 2002Co-Authors: Robert P Carty, Matthew R Pincus, Harold A. ScheragaAbstract:In the initial stages of the oxidative folding of both bovine Pancreatic Ribonuclease A (RNase A) and a 58-72 fragment thereof from the fully reduced, denatured state, the 65-72 correctly paired disulfide bond forms in preponderance over the incorrectly paired 58-65 disulfide bond. Since both disulfide-bonded loops contain the same number of amino acid residues, the question arises as to whether the native pairing results from interactions within the 58-72 segment that lead to a nativelike structure even in its fully reduced form. To answer this question, the chain buildup procedure, based on ECEPP, including a solvation treatment, was used to generate the low-energy structures for the 58-72 RNase segment, beginning with residue 72 and building back to residue 58; in this fragment, all three Cys residues (at positions 58, 65, and 72) initially exist in the reduced (CysH) state. After the open-chain energy minima of the 65-72 peptide were generated, these conformations were allowed to form the 65-72 disulfide bond, and the energies of the resulting oxidized conformations were reminimized and rehydrated. The global minimum of the loop-closed 65-72 structure and many of the low-lying loop-closed minima could be superimposed on the energy-minimized X-ray structure for residues 65-72. The low-energy structures for the full open chain 58-72 peptide were then computed and were allowed to form disulfide bonds either between residues 65 and 72 (native) or between residues 58 and 65 (non-native), and their energies were reminimized and rehydrated in the loop-closed state. Although the overall fold of the 65-72 loop-closed global minimum was the same as for the energy-minimized X-ray structure of these residues, the overall rms deviation was 3.9 A because of local deviations among residues 58-64. In contrast, the 65-72 segment of the global minimum of the 58-72 fragment could be superimposed on the corresponding residues of the energy-minimized X-ray structure. The lowest-energy structure for the 58-65 non-native paired 58-72 sequence was 6 kcal/mol higher in energy than that for the 58-72 peptide with the 65-72 disulfide bond formed. These results suggest that the native pairing of the 65-72 peptide arises from energetic determinants (adoption of left-handed single-residue conformations by Gly 68, and side chain interactions involving Gln 69) contained within this peptide sequence.
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oxidative folding of bovine Pancreatic Ribonuclease a insight into the overall catalysis of the refolding pathway by phosphate
Journal of Protein Chemistry, 2002Co-Authors: Lawrence K Low, Hangcheol Shin, Harold A. ScheragaAbstract:The effects of the strong stabilizing anion, phosphate, on the oxidative folding of bovine Pancreatic Ribonuclease A were examined. Phosphate was found to catalyze several steps involved in the oxidative folding process at pH 8.0 and 25 degrees C, resulting in an increase in the rate of pre-equilibration of unstructured species on the folding pathway. In the presence of 400 mM phosphate, the overall increase in the rate of regeneration of native protein was caused primarily by the increased formation and stabilization of tertiary structure in the nativelike intermediates, des-[40-95] and des-[65-72], involved in the rate-determining step. Based on the regeneration of native protein and the stability of Cys--> Ala substituted mutant analogs of the des-species, (C40A, C95A) and (C65A, C72A), it is suggested that the primary role of phosphate is to catalyze the overall regeneration of native protein through nonspecific electrostatic and hydrogen-bonding effects on the protein and solvent.
Janendra K. Batra - One of the best experts on this subject based on the ideXlab platform.
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a Ribonuclease inhibitor resistant dimer of human Pancreatic Ribonuclease displays specific antitumor activity
International Journal of Biological Macromolecules, 2018Co-Authors: Ayush Attery, Prajna Tripathi, Janendra K. BatraAbstract:Abstract Human Pancreatic Ribonuclease (HPR) and bovine seminal Ribonuclease (BS-RNase) are members of the RNase A superfamily. HPR is monomeric, whereas BS-RNase is dimeric. BS-RNase has strong antitumor and cytotoxic activities. However, HPR lacks cytotoxic activity as it is inactivated by intracellular cytosolic Ribonuclease inhibitor (RI). Earlier, an RI-resistant cytotoxic variant of HPR, termed HPR-KNE was generated which contained three residues Lys7, Asn71 and Glu111 of HPR, known to interact with RI, mutated to alanine. In this study, we have engineered HPR to develop two dimeric RI-resistant molecules having anti-tumor activity. By incorporating two cysteines in HPR and HPR-KNE, we generated disulfide linked dimeric HPR, and a dimer of HPR-KNE, termed as HPR-D and HPR-KNE-D respectively. HPR-KNE-D was resistant towards inhibition by RI, and was found to be highly toxic to a variety of cells. On J774A.1 cells HPR-KNE-D was >375-fold more cytotoxic than HPR, and 15-fold more toxic than HPR-D. Further, on U373 cells HPR-KNE-D was >65-fold more cytotoxic than HPR, and 9-fold more toxic than HPR-D. The study demonstrates that combining dimerization and RI-resistance results in providing potent anti-tumor activity to HPR. The cytotoxic variants of HPR will be useful in designing protein therapeutics with low immunogenicity.
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functional role of glutamine 28 and arginine 39 in double stranded rna cleavage by human Pancreatic Ribonuclease
PLOS ONE, 2011Co-Authors: Md Tabish Rehman, Punyatirtha Dey, Md Imtaiyaz Hassan, Faizan Ahmad, Janendra K. BatraAbstract:Human Pancreatic Ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR.
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interaction of human Pancreatic Ribonuclease with human Ribonuclease inhibitor generation of inhibitor resistant cytotoxic variants
Journal of Biological Chemistry, 2001Co-Authors: Deepak Gaur, Srividya Swaminathan, Janendra K. BatraAbstract:Abstract Mammalian Ribonucleases interact very strongly with the intracellular Ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian Ribonucleases are protected from their cytotoxic action by the intracellular inhibition of Ribonucleases by RI. Human Pancreatic Ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human Ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human Pancreatic Ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.
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interaction of human Pancreatic Ribonuclease with human Ribonuclease inhibitor generation of inhibitor resistant cytotoxic variants
Journal of Biological Chemistry, 2001Co-Authors: Deepak Gaur, Srividya Swaminathan, Janendra K. BatraAbstract:Abstract Mammalian Ribonucleases interact very strongly with the intracellular Ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian Ribonucleases are protected from their cytotoxic action by the intracellular inhibition of Ribonucleases by RI. Human Pancreatic Ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human Ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human Pancreatic Ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.
Maria Vilanova - One of the best experts on this subject based on the ideXlab platform.
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a nuclear directed human Pancreatic Ribonuclease pe5 targets the metabolic phenotype of cancer cells
Oncotarget, 2016Co-Authors: Anna Vert, Antoni Benito, Marc Ribo, Jessica Castro, Maria VilanovaAbstract:// Anna Vert 1, 2 , Jessica Castro 1, 2 , Marc Ribo 1, 2 , Antoni Benito 1, 2 , Maria Vilanova 1, 2 1 Laboratori d'Enginyeria de Proteines, Departament de Biologia, Facultat de Ciencies, Universitat de Girona, Girona, Spain 2 Institut d’Investigacio Biomedica de Girona Josep Trueta, (IdIBGi), Girona, Spain Correspondence to: Maria Vilanova, email: maria.vilanova@udg.edu Antoni Benito, email: antoni.benito@udg.edu Keywords: antitumor drug, human Pancreatic Ribonuclease, metabolism of cancer cells, microarray profiling, tumor cell death Received: December 18, 2015 Accepted: February 11, 2016 Published: February 22, 2016 ABSTRACT Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted Ribonuclease family are not cytotoxic because they are not able to evade the cytosolic Ribonuclease inhibitor. We previously engineered the human Pancreatic Ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21 WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.
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the nuclear transport capacity of a human Pancreatic Ribonuclease variant is critical for its cytotoxicity
Investigational New Drugs, 2011Co-Authors: Pere Tubert, Antoni Benito, Marc Ribo, Montserrat Rodriguez, Maria VilanovaAbstract:We have previously described a human Pancreatic-Ribonuclease variant, named PE5, which carries a non-contiguous extended bipartite nuclear localization signal. This signal comprises residues from at least three regions of the protein. We postulated that the introduction of this signal in the Ribonuclease provides it with cytotoxic activity because although the variant poorly evades the Ribonuclease inhibitor in vitro, it is routed to the nucleus, which is devoid of the inhibitor. In this work, we have investigated the relationship between the cytotoxicity produced by PE5 and its ability to reach the nucleus. First, we show that this enzyme, when incubated with HeLa cells, specifically cleaves nuclear RNA while it leaves cytoplasmic RNA unaffected. On the other hand, we have created new variants in which the residues of the nuclear localization signal that are important for the nuclear transport have been replaced. As expected, the individual changes produce a significant decrease in the cytotoxicity of the resulting variants. We conclude that the nuclear transport of PE5 is critical for its cytotoxicity. Therefore, routing a Ribonuclease to the nucleus is an alternative strategy to endow it with cytotoxic activity.
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a nuclear localization sequence endows human Pancreatic Ribonuclease with cytotoxic activity
Biochemistry, 2004Co-Authors: Montserrat Bosch, Antoni Benito, Marc Ribo, Teresa Puig, Bruno Beaumelle, Maria VilanovaAbstract:Some members of the Ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human Pancreatic Ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic Ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human Pancreatic Ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human Pancreatic Ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5−15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization ...
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three dimensional structure of a human Pancreatic Ribonuclease variant a step forward in the design of cytotoxic Ribonucleases
Journal of Molecular Biology, 2000Co-Authors: Joan Pous, Antoni Benito, Marc Ribo, Maria Vilanova, Albert Canals, Simon Terzyan, Alicia Guasch, Miquel CollAbstract:Abstract We have determined the crystal structure of a human Pancreatic Ribonuclease or RNase 1 variant at 1.65 A resolution. Five residues in the N-terminal region were substituted by the corresponding amino acids of the bovine seminal RNase. In addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in improving the expression of this enzyme as a recombinant protein and in achieving its crystallisation. The determination of the crystal structure revealed the characteristic RNase fold including a V-shaped β-sheet and three α-helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that of its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human Ribonuclease with the Ribonuclease inhibitor, together with inhibition assays, indicate that, in contrast to RNase A, the modification of the loop β4β5 is not enough to avoid inhibition. This study represents the first crystallographic approach to the human enzyme, and should constitute an invaluable tool for the design of Ribonuclease variants with acquired cytotoxic properties.
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bovine Pancreatic Ribonuclease a as a model of an enzyme with multiple substrate binding sites
Biochimica et Biophysica Acta, 1995Co-Authors: Victoria M Nogues, Maria Vilanova, Claudi M CuchilloAbstract:Abstract Bovine Pancreatic Ribonuclease A is an enzyme that catalyses the depolymerization of RNA. This process involves the interaction of the enzyme with the alignment on the substrate in the active site and its correct alignment on the surface of the enzyme through multiple binding subsites that essentially recognize the negatively charged phosphate groups of the substrate. The enzyme shows a strong specificity for pyrimidine bases at the 3′-position of the phosphodiester bond that is cleaved and a preference for purine bases at the 5′-position and, probably, for guanine at the next position. On the other hand, the enzyme shows a clear preference for polynucleotide substrates over oligonucleotides. In this review the contributions to the catalytic mechanism of some amino-acid residues that are located at non catalytic binding subsites are analysed.
Ronald T. Raines - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of human Pancreatic Ribonuclease by the human Ribonuclease inhibitor protein.
Journal of molecular biology, 2007Co-Authors: R. Jeremy Johnson, J G Mccoy, G N Phillips, Craig A. Bingman, Ronald T. RainesAbstract:The Ribonuclease inhibitor protein (RI) binds to members of the bovine Pancreatic Ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human Pancreatic Ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered Ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human Ribonucleases as chemotherapeutic agents.
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inhibition of human Pancreatic Ribonuclease by the human Ribonuclease inhibitor protein
Journal of Molecular Biology, 2005Co-Authors: Jeremy R Johnson, J G Mccoy, G N Phillips, Craig A. Bingman, Ronald T. RainesAbstract:The Ribonuclease inhibitor protein (RI) binds to members of the bovine Pancreatic Ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human Pancreatic Ribonuclease (RNase 1). The structure of the crystalline hRI {center_dot} RNase 1 complex was determined at a resolution of 1.95 {angstrom}, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI {center_dot} RNase 1 complex was found to be extremely stable (t{sub 1/2} = 81 days; K{sub d} = 2.9 x 10{sup -16}). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed 'electrostatic targeting residues'. Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1more » that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10{sup 3}-fold lower association rate for complex formation and 5.9 x 10{sup 9}-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered Ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human Ribonucleases as chemotherapeutic agents.« less
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endowing human Pancreatic Ribonuclease with toxicity for cancer cells
Journal of Biological Chemistry, 2001Co-Authors: Peter A Leland, Kristine E Staniszewski, Byungmoon Kim, Ronald T. RainesAbstract:Abstract Onconase® is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human Pancreatic Ribonuclease (RNase 1) is homologous to Onconase® but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but >103-fold less affinity for the endogenous cytosolic Ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC50 value is only 8-fold greater than that of Onconase®. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
Antoni Benito - One of the best experts on this subject based on the ideXlab platform.
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a nuclear directed human Pancreatic Ribonuclease pe5 targets the metabolic phenotype of cancer cells
Oncotarget, 2016Co-Authors: Anna Vert, Antoni Benito, Marc Ribo, Jessica Castro, Maria VilanovaAbstract:// Anna Vert 1, 2 , Jessica Castro 1, 2 , Marc Ribo 1, 2 , Antoni Benito 1, 2 , Maria Vilanova 1, 2 1 Laboratori d'Enginyeria de Proteines, Departament de Biologia, Facultat de Ciencies, Universitat de Girona, Girona, Spain 2 Institut d’Investigacio Biomedica de Girona Josep Trueta, (IdIBGi), Girona, Spain Correspondence to: Maria Vilanova, email: maria.vilanova@udg.edu Antoni Benito, email: antoni.benito@udg.edu Keywords: antitumor drug, human Pancreatic Ribonuclease, metabolism of cancer cells, microarray profiling, tumor cell death Received: December 18, 2015 Accepted: February 11, 2016 Published: February 22, 2016 ABSTRACT Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted Ribonuclease family are not cytotoxic because they are not able to evade the cytosolic Ribonuclease inhibitor. We previously engineered the human Pancreatic Ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21 WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells.
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the nuclear transport capacity of a human Pancreatic Ribonuclease variant is critical for its cytotoxicity
Investigational New Drugs, 2011Co-Authors: Pere Tubert, Antoni Benito, Marc Ribo, Montserrat Rodriguez, Maria VilanovaAbstract:We have previously described a human Pancreatic-Ribonuclease variant, named PE5, which carries a non-contiguous extended bipartite nuclear localization signal. This signal comprises residues from at least three regions of the protein. We postulated that the introduction of this signal in the Ribonuclease provides it with cytotoxic activity because although the variant poorly evades the Ribonuclease inhibitor in vitro, it is routed to the nucleus, which is devoid of the inhibitor. In this work, we have investigated the relationship between the cytotoxicity produced by PE5 and its ability to reach the nucleus. First, we show that this enzyme, when incubated with HeLa cells, specifically cleaves nuclear RNA while it leaves cytoplasmic RNA unaffected. On the other hand, we have created new variants in which the residues of the nuclear localization signal that are important for the nuclear transport have been replaced. As expected, the individual changes produce a significant decrease in the cytotoxicity of the resulting variants. We conclude that the nuclear transport of PE5 is critical for its cytotoxicity. Therefore, routing a Ribonuclease to the nucleus is an alternative strategy to endow it with cytotoxic activity.
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a nuclear localization sequence endows human Pancreatic Ribonuclease with cytotoxic activity
Biochemistry, 2004Co-Authors: Montserrat Bosch, Antoni Benito, Marc Ribo, Teresa Puig, Bruno Beaumelle, Maria VilanovaAbstract:Some members of the Ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human Pancreatic Ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic Ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human Pancreatic Ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human Pancreatic Ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5−15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization ...
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three dimensional structure of a human Pancreatic Ribonuclease variant a step forward in the design of cytotoxic Ribonucleases
Journal of Molecular Biology, 2000Co-Authors: Joan Pous, Antoni Benito, Marc Ribo, Maria Vilanova, Albert Canals, Simon Terzyan, Alicia Guasch, Miquel CollAbstract:Abstract We have determined the crystal structure of a human Pancreatic Ribonuclease or RNase 1 variant at 1.65 A resolution. Five residues in the N-terminal region were substituted by the corresponding amino acids of the bovine seminal RNase. In addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in improving the expression of this enzyme as a recombinant protein and in achieving its crystallisation. The determination of the crystal structure revealed the characteristic RNase fold including a V-shaped β-sheet and three α-helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that of its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human Ribonuclease with the Ribonuclease inhibitor, together with inhibition assays, indicate that, in contrast to RNase A, the modification of the loop β4β5 is not enough to avoid inhibition. This study represents the first crystallographic approach to the human enzyme, and should constitute an invaluable tool for the design of Ribonuclease variants with acquired cytotoxic properties.
