The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.     

Interaction of human placental ribonuclease with placental ribonuclease inhibitor

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.     

The cytosolic ribonuclease inhibitor contributes to intracellular redox homeostasis

The hypothesis that the cytosolic RNase inhibitor (cRI) has a role in the protection of the cellular redox homeostasis was investigated testing the effects of oxidants and anti-oxidants on normal, primary endothelial HUVE cells, and malignant HeLa cells, before and after their engineering into cRI-deprived cells. We found that cRI plays an important, possibly a key, physiological role in the protection of cells from redox stress, as demonstrated by decreased GSH levels as well as increased oxidant-induced DNA damage in cRI deprived cells.     

Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the complex in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 degrees C, this value is 1.3 X 10(-7) s-1 (t1/2 congruent to 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 X 10(-16) M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 X 10(-5) s-1 (t1/2 = 13 h), and the Ki value is 4.4 X 10(-14) M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2'-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2'-phosphate decrease the association rate in a manner consistent with a competitive mode of inhibition.     

Inhibition of human pancreatic ribonuclease by the human ribonuclease inhibitor protein

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.     

Antitumor effects of human ribonuclease inhibitor gene transfected on B16 melanoma cells

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

Variants of ribonuclease inhibitor that resist oxidation

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.     

Primary structure of human placental ribonuclease inhibitor

The primary structure of the human placental ribonuclease inhibitor (PRI), a tight-binding inhibitor of angiogenin and pancreatic ribonucleases, has been determined from the cDNA. The sequence of the mature protein is composed of 460 amino acids, yielding a molecular mass of 49,847 g/mol. Peptides comprising 92% of the predicted sequence were isolated from a tryptic digest of PRI, and direct sequence information obtained for 65% of the molecule agreed at all positions with the sequence predicted from the cDNA. The amino acid sequence of PRI contains seven direct internal repeat units, each 57 amino acids in length. These repeat units comprise 87% of the molecule. The average degree of identity between any two is 39%. A region within each repeat unit displays similarity to tandem, leucine-rich repeats found in six other proteins. Modification of PRI with iodoacetic acid, p-(hydroxymercuri)benzoate, and 5,5'-dithiobis(2-nitrobenzoic acid) reveals that at least 30 of the 32 cysteine residues of PRI are in the reduced form.     

Fluorescence assay for the binding of ribonuclease A to the ribonuclease inhibitor protein

Ribonuclease A (RNase A) and the ribonuclease inhibitor protein (RI) form one of the tightest known protein-protein complexes. RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells. Herein, a new and facile assay is described for measuring the equilibrium dissociation constant (K(d)) and dissociation rate constant (k(d)) for complexes of RI and RNase A. This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI. To allow time for equilibration, the assay is most readily applied to those complexes with K(d) values in the nanomolar range or higher. Using this assay, the value of K(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 +/- 0.03 nM. In addition, the value of K(d) was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 +/- 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A. Finally, the value of k(d) for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 +/- 0.4) x 10(-3) s(-1) by monitoring the increase in fluorescence intensity upon dissociation. This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.     

Superadditive and subadditive effects of "hot spot" mutations within the interfaces of placental ribonuclease inhibitor with angiogenin and ribonuclease A.

Previous single-site mutagenesis studies on the complexes of ribonuclease inhibitor (RI) with angiogenin (Ang) and RNase A suggested that in both cases a substantial fraction of the binding energy is concentrated within one small part of the crystallographically observed interface, involving RI residues 434-438. Such energetic "hot spots" are common in protein-protein complexes, but their physical meaning is generally unclear. Here we have investigated this question by examining the detailed interactions within the RI.ligand hot spots and the extent to which they function independently. The effects of Phe versus Ala substitutions show that the key residue Tyr434 interacts with both ligands primarily through its phenyl ring; for Tyr437, the OH group forms the important contacts with RNase A, whereas the phenyl group interacts with Ang. Kinetic characterization of complexes containing multiple substitutions reveals striking, but distinctive, cooperativity in the interactions of RI with the two ligands. The losses in binding energy for the RNase complex associated with replacements of Tyr434 and Asp435, and Tyr434 and Tyr437, are markedly less than additive (i.e., by 2.4 and 1.3 kcal/mol, respectively). In contrast, the energetic effects of the 434 and 435, and 434 and 437, substitution pairs on binding of Ang are fully additive and 2.5 kcal/mol beyond additive, respectively. Superadditivities (0.9-2.4 kcal/mol) are also observed for several multisite replacements involving these inhibitor residues and two Ang residues, Arg5 and Lys40, from this part of the interface. Consequently, the decreases in binding energy for some triple-variant complexes are as large as 8.5-10.1 kcal/mol (compared to a total DeltaG of -21.0 kcal/mol for the wild-type complex). Potential explanations for these functional couplings, many of which occur over distances of >13 A and are not mediated by direct or triangulated contacts, are proposed. These findings show that the basis for the generation of hot spots can be complex, and that these sites can assume significantly more (as with Ang) or less (as with RNase) importance than indicated from the effects of single-site mutations.     

Inactivation of ribonuclease inhibitor by thiol-disulfide exchange.

Porcine ribonuclease inhibitor (RI) contains 30 1/2-cystinyl residues, all of which occur in the reduced form. Reaction of the native protein with 5,5'-dithiobis (2-nitrobenzoic acid) resulted in the release of 30 mol of the product 5-mercapto-2-nitrobenzoate, and the loss of the RNase inhibitory activity. A linear relationship between the degree of modification and inactivation was observed. The rate of modification was greatly increased in the presence of 6 M guanidinium HCl. Reaction with substoichiometric amounts of 5,5'-dithiobis(2-nitrobenzoic acid) was found to yield a mixture of fully reduced active molecules, and fully oxidized inactive ones, but no partially oxidized forms were detected. This suggests that an "all-or-none" type of modification and inactivation took place. All 1/2-cystinyl residues in the inactive, monomeric inhibitor had formed disulfide bridges, judged by the absence of either free thiol groups or mixed disulfides with 5-mercapto-2-nitrobenzoate. This fully disulfide-cross-linked molecule had an open conformation compared to the native one, as shown by gel filtration and limited proteolysis. Reaction of phenylarsinoxide with vicinal dithiols yields products that are much more stable than those with monothiols. Titration of RI with this reagent yielded complete inactivation at a reagent/thiol ratio of 0.5. Taken together, these observations suggest that the thiol groups in RI have a diminished reactivity due to three-dimensional constraints. After the initial modification of a small number of thiol groups, a conformational change occurs which causes an increase in reactivity of the remaining thiols. The thiol groups are situated close enough together to permit the formation of 15 disulfide bridges in the inactive molecule.     

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants

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.     

Analysis of the interactions of ribonuclease inhibitor with kanamycin

The interaction of ribonuclease inhibitor (RI) with kanamycin was studied by molecular modeling. The preliminary binding model was constructed using the Affinity module of the Insight II molecular modeling program and the key residues involved in the combination of RI binding to kanamycin were determined. Meanwhile, we determined relevant surface characteristics determining the interaction behavior. The modeling results indicated that electrostatic interactions and H-bond forces may work as major factors for the molecular interaction between kanamycin and RI. The above results are useful for elucidating the molecular principles upon which the selectivity of a kanamycin is based. The quartz-crystal microbalance (QCM) is a new method usually used to monitor the binding function of macromolecules with samples online in a flow-injection analysis (FIA) system. The experimental results demonstrate that kanamycin has an extraordinary affinity to the basic protein RI, and our result is consistent with the molecular modeling results. These principles can in turn be used to study the molecular recognition mechanism and design a mimic of kanamycin for the development of new RI binders.Figure Proposed binding model of kanamycin to RI obtained by the computer-aided docking method and optimized with molecular mechanics with the CVFF forcefield.