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N Russo

Publications and source records attributed to N Russo.

At least 19 recordsLinked to original sources
PubMed2000

In vitro evolution of a dimeric variant of human pancreatic ribonuclease.

Site-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of >280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N-terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 degrees C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 microM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d) > 73 mM). Cross-linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers.

Amino Acid Sequence↗
PubMed2000

The two dimeric forms of RNase A.

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868-3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437-3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.

Animals↗
PubMed2000

Gas-phase metal ion (Li+, Na+, Cu+) affinities of glycine and alanine.

The gas-phase metal affinities of glycine and alanine for Li+, Na+ and Cu+ ions have been determined theoretically employing the hybrid B3LYP exchange-correlation functional and using extended basis sets. All computations indicate that the metal ion affinity (MIA) decreases on going from Cu+ to Li+ and Na+ for both the considered amino acids. The absolute MIA values are close to the experimental counterparts with the exception of lithium for which a deviation of about 7 kcal/mol at the B3LYP level is obtained. The optimized structures indicate that Li+, Na+ and Cu+ prefer a bidentate coordination, bonding with both nitrogen and oxygen atoms of amino acids.

Alanine↗
PubMed2000

Semiempirical molecular modeling into quercetin reactive site: structural, conformational, and electronic features.

The conformational behavior, molecular geometry and electronic structure of quercetin were investigated using the semiempirical AM1 and PM3 methods. Results reveal that quercetin has a nonplanar molecular structure, with cross-conjugation occurring at the C ring. Calculations were also performed for quercetin radical species at the OH groups, showing the presence of three radicals in a narrow range of energy. An interpretation of the antioxidative process mechanism, exerted by quercetin as a free radical scavenger, relies on two isoenergetic radicals with extended electronic delocalization between adjacent rings, also having cross-conjugated systems and being affected by the experimental environment influencing their relative order.

Electrons↗
PubMed1999

Toward rational design of ribonuclease inhibitors: high-resolution crystal structure of a ribonuclease A complex with a potent 3',5'-pyrophosphate-linked dinucleotide inhibitor.

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

Adenosine Diphosphate↗
PubMed1999

Potent inhibition of mammalian ribonucleases by 3', 5'-pyrophosphate-linked nucleotides.

Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' --> 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1-2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes.

Adenosine Diphosphate↗
PubMed1999

Theoretical calculations of glycine and alanine gas-phase acidities.

The gas-phase acidities of glycine and alanine were determined by using a variety of high level theoretical methods to establish which of these would give the best results with accessible computational efforts. MP2, MP4, QCISD, G2 ab initio procedures, hybrid Becke3-LYP (B3LYP) and gradient corrected Becke-Perdew (BP) and Perdew-Wang and Perdew (PWP) nonlocal density functionals were used for the calculations. A maximum deviation of approximately 13 and 18 kJ/mol from experimental data was observed for the computed delta Hacid and delta Gacid values, respectively. The best result was obtained at G2 level, but comparable reliability was reached when the considerably less time consuming B3LYP, BP, and PWP density functional approaches were employed.

Alanine↗
PubMed1998

Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.

Base Sequence↗
PubMed1997

Crystal structures of ribonuclease A complexes with 5'-diphosphoadenosine 3'-phosphate and 5'-diphosphoadenosine 2'-phosphate at 1.7 A resolution.

High-resolution (1.7 A) crystal structures have been determined for bovine pancreatic ribonuclease A (RNase A) complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) and 5'-diphosphoadenosine 2'-phosphate (ppA-2'-p), as well as for a native structure refined to 2.0 A. These nucleotide phosphates are the two most potent inhibitors of RNase A reported so far, with Ki values of 240 and 520 nM, respectively. The binding modes and conformations of ppA-3'-p and ppA-2'-p were found to differ markedly from those anticipated on the basis of earlier structures of RNase A complexes. The key difference is that the 5'-beta-phosphate rather than the 5'-alpha-phosphate of each inhibitor occupies the P1 phosphate binding site. As a consequence, the ribose moieties of the two nucleotides are shifted by approximately 2 A compared to the positions of their counterparts in earlier complexes, and the adenine rings are rotated into unusual syn conformations. Thus, the six-membered and five-membered rings of both adenines are reversed with respect to the others but nonetheless engage in extensive interactions with the residues that form the B2 purine binding site of RNase A. Despite the close structural similarity of the two inhibitors, the puckers of their furanose rings are different: C2'-endo and C3'-endo, respectively. Moreover, their 5'-alpha-phosphates and 3'(2')-monophosphates interact with largely different sets of RNase residues. The results of this crystallographic study emphasize the difficulties inherent in qualitative modeling of protein-inhibitor interactions and the compelling reasons for high-resolution structural studies in which quantitative design of improved inhibitors was enabled. The structures presented here provide a promising starting point for the rational design of tight-binding RNase inhibitors, which may be used as therapeutic agents in restraining the ribonucleolytic activities of RNase homologues such as angiogenin, eosinophil-derived neurotoxin, and eosinophil cationic protein.

Adenosine Diphosphate↗
PubMed1997

5'-Diphosphoadenosine 3'-phosphate is a potent inhibitor of bovine pancreatic ribonuclease A.

As a first step toward the development of stable, selective, and potent inhibitors of those members of the pancreatic RNase superfamily that induce biological responses, we have focussed on low molecular weight compounds and studied their interactions with the active-site of bovine pancreatic ribonuclease A (RNase A). A new inhibitor is described, 5'-diphosphoadenosine 3'-phosphate, which binds to RNase A more tightly than any previous low molecular weight compound: its Ki value of 1.3 microM at pH 7 is 8-fold lower than that for uridine-vanadate, a transition-state analog, and 110-fold lower than that for 2'-CMP, one of the best-characterized RNase A ligands. The new inhibitor is found to contact RNase A residues that are conserved in several homologous mammalian RNases and hence should be able to serve as a basis for the design of even tighter-binding inhibitors of these enzymes.

Adenine Nucleotides↗
PubMed1997

Antiaggregatory effects of picotamide in long-term treatment: a 2-year, double-blind placebo-controlled trial.

The ex vivo antiaggregatory activity of picotamide, a dual antithromboxane agent, was assessed to find whether it was maintained in long-term treatment. In a double-blind, placebo-controlled 2-year study, 50 type 2 diabetic patients (35 men and 15 women; mean age 66 +/- 5 years) were enrolled and randomly given picotamide, 300 mg t.i.d. or the corresponding placebo. Platelet aggregation studies were performed at baseline and after 1, 3, 6, 12, 18 and 24 months. Compliance to the treatment was assessed by pill count at each visit. Forty-nine patients concluded the study. Starting from month 1, compared with placebo, picotamide-treated patients showed a significant inhibition of agonist-induced (ADP, arachidonic acid and collagen) platelet aggregation (-41%). The antiaggregatory effect was maintained throughout the study. At month 24, in the picotamide group, platelet aggregation was significantly lower compared with placebo (-30%). After 24 months of treatment, 20 out of 23 (86%) picotamide-treated patients showed a significant inhibition of platelet aggregation, whereas the remaining three patients had a normal platelet response. During the study, 12 patients suffered from thrombotic events of death: nine in the placebo group and three in the picotamide group, respectively. It was concluded that picotamide maintains its antiaggregatory effect, in long-term treatment, in more than 85% of patients.

Aged↗
PubMed1996

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

Amino Acid Sequence↗
PubMed1996

A combined kinetic and modeling study of the catalytic center subsites of human angiogenin.

Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B1 pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B1. Although the B2 site that preferentially binds purines on the 3' side of B1 is also weak, its associated phosphate subsites make substantial contributions: both 3',5'-ADP and 5'-ADP have Ki values 6-fold lower than for 5'-AMP, and adding a 3'-phosphate to the substrate CpA increases Kcat/Km by 9-fold. Thus Ang has a functional P2 site on the 3' side of B2 and a site for a second phosphate on the 5' side of B2. Modeling of an Ang-d(ApTpApA) complex suggested that Arg-5 forms part of the P2 site and that a 2'-phosphate might bind more tightly than a 3'-phosphate. Both predictions were confirmed kinetically. The subsite map obtained by this combined approach indicated that 5'-diphosphoadenosine 2'-phosphate might be a more potent inhibitor than any of the nucleotides tested thus far. Indeed, its Ki value of 150 microM is 50-fold lower than that for the best nucleotide previously reported and 400-fold lower than the Km for the best dinucleotide substrate. This compound may serve as a suitable starting point for the eventual design of tight-binding inhibitors of Ang as antiangiogenic agents for human therapy.

Adenosine Diphosphate↗
PubMed1995

Conformational behaviour of the antineoplastic peptide dolastatin-10 and of two mutated derivatives.

The three-dimensional structure of dolastatin-10, an extremely potent cytostatic and antineoplastic peptide extracted from the mollusc Dolabella auricularia, has not yet been fully characterized in an experimental way. By means of a systematic conformational search of the natural peptide and of two mutated analogs, carried out both in vacuo and in aqueous solution, the present work allows to obtain insights into the conformational preferences of this remarkable compound. In addition, the ability to form intra- and intermolecular H-bonds as a function both of the sequence and of the conformation is discussed. The search for the best molecular conformations has been carried out using a molecular mechanics approach, based on the CVFF potential. Dolastatin-10 contains some unusual amino acids for which no experimental structural data are available. In order to check the reliability of the CVFF potential in predicting structures of such nonconventional amino acids, geometry optimizations have been carried out using the ab initio Hartree-Fock procedure. The CVFF parameterization is found to be adequate also for nonconventional amino acids.

Amino Acids↗
PubMed1995

Effects of picotamide, an antithromboxane agent, on carotid atherosclerotic evolution. A two-year, double-blind, placebo-controlled study in diabetic patients.

BACKGROUND AND PURPOSE: We assessed the effects of long-term treatment with picotamide, an antiplatelet agent with dual antithromboxane activity, on the evolution of early asymptomatic carotid atherosclerotic lesions in diabetic patients. METHODS: In a double-blind, placebo-controlled, 2-year study, 50 type II normotensive diabetic patients (35 men; mean age, 66 +/- 5 years) with asymptomatic mild or moderate nonstenotic (< 50%) carotid atherosclerotic lesions and negative history of cerebrovascular ischemic events were enrolled and randomly given picotamide (300 mg TID) or the corresponding placebo. A high-resolution, real-time B-scan echographic assessment of carotid arteries was performed at baseline and after 1, 3, 6, 12, 18, and 24 months of double-blind treatment. Prevalence and evolutionary trends of carotid atherosclerotic lesions (number per patient and mean stenosis expressed as percent) were considered as efficacy primary end points. RESULTS: At baseline, mean +/- SD numbers of carotid atherosclerotic lesions per patient were 2.7 +/- 1.8 and 2.2 +/- 1.2 in the picotamide and placebo groups, respectively. Mean +/- SD percent stenosis was 25.3 +/- 7% in the picotamide group and 27.3 +/- 6% in the placebo group. Forty-nine patients completed the study. At month 24, the placebo group (n = 24) showed a significant progression in number of carotid atherosclerotic lesions (3.04 +/- 1.8; P < .02 versus baseline) and in mean percent stenosis (35 +/- 17%; 95% confidence interval, 33% to 37%; P < .01 versus baseline). In the picotamide group (n = 25), mean number of carotid atherosclerotic lesions (2.7 +/- 1.6) and percent stenosis (26 +/- 9%; 95% confidence interval, 24.8% to 27.2%) remained unchanged. At month 24, compared with randomized placebo, lesion numbers (P < .03) and percent stenosis (P < .01) in the picotamide group were significantly lower. During the study, 12 patients experienced major or minor ischemic vascular events (9 in the placebo group and 3 in the picotamide group; P = .07). CONCLUSIONS: In diabetic patients compared with patients receiving placebo, long-term treatment with picotamide can slow the evolution of early carotid atherosclerotic lesions, inhibiting progression of plaque number and growth.

Administration, Oral↗