Ribonuclease Molecule Research Articles

Evaluation of RNase therapy in systemic lupus erythematosus: a randomised phase 2a clinical trial of RSLV-132

BackgroundCirculating, extracellular RNA is the primary trigger of type I interferon in systemic lupus erythematosus (SLE), and interferon is known to play a central pathogenic role in the disease. RSLV-132...

Ribonuclease as Anticancer Therapeutics

Ribonucleases (RNases) are small molecules which are highly cytotoxic in nature. They catalyse the degradation of RNA into smaller molecules rapidly in an unprotected environment. The cytotoxic properties of RNases include degradation of RNA leading to blockage of protein synthesis in malignant cells and inducing the apoptosis response. Cytotoxicity of RNases is determined by catalytic activity, stability, non-selective nature of inhibitors, positive charge on molecule and internalization. Onconase, BS-RNase and other RNases exert cytotoxic activity on cancer cells selectively by involving different cellular pathways and/ or enhance the cytotoxicity by mutation. A general mechanism of the cytotoxic activity of RNases includes the interaction of the enzyme with the cellular membrane by non-specific interactions mediated by Coulombic forces, internalization by endocytosis, translocation to the cytosol, degradation of ribonucleic acid and subsequent cell death by activation of caspase-dependent mechanisms, low molecular weight compounds or alteration in protein and NF-κB signal pathway. But still it is unclear that which of these mechanisms is most potent, common and causes cell death in cancerous cells. The problem related to ribonuclease inhibitor (RI) has not fully elucidated. This article looks at the cellular pathways of RNases and mechanism of their cytotoxicity towards the malignant cells which makes RNase as a strong candidate to be considered as chemotherapeutic or antitumor drug. Some of the prominent approaches to exploit RNase molecule as an anticancer therapeutic agent have been discussed.

Balnase, a New Dimer-Forming Ribonuclease from Bacillus altitudinis

Current cancer treatments still remain ineffective and cause side effects which ruinously affect healthy cells. Among new promising anticancer drugs, special attention is paid to the ribonucleases (RNases) which possess selective cytotoxicity against malignant cells. It is found that besides enzymatic activity conformational state of RNase molecules plays a role in the anticancer activity. Balnase, a new RNase from Bacillus altitudinis, is a close homolog of Bacillus pumilus RNase (binase) which selectively kills malignant cells expressing oncogenes KIT, ras, AML1/ETO, and FLT3. Earlier, homogeneous sample of balnase was obtained. Here, we have characterized structural organization of balnase. It was shown that it is a natural dimer. The enzyme has the same conformational state as binase but balnase dimers are less stable.

Targeting Divalent Metal Ions at the Active Site of the HIV-1 RNase H Domain: NMR Studies on the Interactions of Divalent Metal Ions with RNase H and Its Inhibitors

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg2+ and Ca2+ bind specifically to the active site of the RNase H domain and two Mg2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.

Effect of Urea and Glycerol on the Adsorption of Ribonuclease A at the Air−Water Interface

This study reports on the influence of nonionic cosolvents on the interfacial structure of ribonuclease A (RNase) adsorbed at the air-water interface. We applied X-ray reflectometry to obtain detailed volume fraction profiles of the adsorbed layers and to follow the effect of glycerol and urea on the adsorbate structure as a function of cosolvent concentration. Under all conditions studied, the adsorbed RNase layer maintains its compact shape, and the adsorbed RNase molecules adopt a flat-on orientation at the interface. Both kosmotropic glycerol and chaotropic urea exert profound effects on the adsorbate: The surface excess decreases linearly with glycerol content and is also reduced at low urea concentration. However, at high urea concentration, parts of the adsorbed layer are dehydrated and become exposed to air. The electron density and volume fraction profiles of the adsorbed protein provide clear evidence that these effects are ruled by different mechanisms.

Aspects of the Cytotoxic Action of Ribonucleases

By virtue of their RNA degrading catalytic activity, ribonucleases are potentially cytotoxic. For the application of these enzymes as therapeutics, however, they have to overcome several obstacles whose interplay is not yet fully understood. Ribonucleases with a basic pI are not only able to interact with the (negatively charged) cellular membrane but they are also distinctively selective for tumor cells. After the (endocytotic) uptake into the cell and release into the cytosol from the endosomes where they have to resist the attack by proteases, they face the cytosolic ribonuclease inhibitor. Only if they are able to evade the tight binding to the inhibitor (or if enough ribonuclease molecules enter the cell to neutralize the inhibitor protein) they are able to attack their target RNA, for which a sufficient ribonucleolytic activity is indispensable. Each of these steps can turn into an insurmountable hurdle spoiling the cytotoxic potential of these enzymes. In the present review I will summarize the status quo of the knowledge on the mechanisms and their interdependence as well as to develop strategies to overcome possible limitations.

Some secretion characteristics of bacterial ribonuclease

The investigation of the effect of some components of the medium on the distribution of the secretory guanyl-specific ribonuclease of Bacillus intermedius (EC 3.1.4.23) among various cell fractions and culture liquid showed that the amount of this enzyme in the culture liquid does not depend on the concentration of calcium ions in the medium (within 1–5 mM). The study of the effect of the amino acid substitutions Trp34Asn and Trp70Asn in the ribonuclease molecule showed that the secretion of ribonuclease depends on the formation rate of its secondary structure. The amino acid substitution Trp34Asn completely inhibits ribonuclease secretion.

Why ribonucleases induce tumor cell death

The characteristics and the possible mechanisms of action of cytotoxic ribonucleases (RNases), promising antitumor drugs, are described. Original experimental data and the results of analysis of recent publications have made it possible to identify the cellular components providing for the selective effects of exogenous RNases on tumor cells, on the one hand, and to estimate the contributions of individual molecular determinants to the enzyme cytotoxicity, on the other hand. The predominant effect of the electric charge of the RNase molecule on the induction of cell death has been demonstrated. The cytotoxic effects of RNases are determined by the catalytic cleavage of accessible RNA, the action of the products of its hydrolysis, and the noncatalytic electrostatic interaction of the exogenous enzyme with cell components. Potential RNase targets in a tumor cell and the role of modulation of calcium-dependent potassium channels and the ras oncogene in RNase-induced cell damage are considered. The effect of cytotoxic RNases on gene expression by affecting RNA interference is discussed.

Conformational subspace in simulation of early‐stage protein folding

A probability calculus was used to simulate the early stages of protein folding in ab initio structure prediction. The probabilities of particular phi and psi angles for each of 20 amino acids as they occur in crystal forms of proteins were used to calculate the amount of information necessary for the occurrence of given phi and psi angles to be predicted. It was found that the amount of information needed to predict phi and psi angles with 5 degrees precision is much higher than the amount of information actually carried by individual amino acids in the polypeptide chain. To handle this problem, a limited conformational space for the preliminary search for optimal polypeptide structure is proposed based on a simplified geometrical model of the polypeptide chain and on the probability calculus. These two models, geometric and probabilistic, based on different sources, yield a common conclusion concerning how a limited conformational space can represent an early stage of polypeptide chain-folding simulation. The ribonuclease molecule was used to test the limited conformational space as a tool for modeling early-stage folding.

Proline versus charge concept for protein stabilization against proteolytic attack.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

Hydrogen Peroxide-induced Structural Alterations of RNase A

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

2-5A-dependent RNase Molecules Dimerize during Activation by 2-5A

2-5A-dependent RNase is an interferon-inducible enzyme that requires 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) for its endoribonuclease activity against single-stranded RNAs. We demonstrate here that recombinant, human 2-5A-dependent RNase forms stable homodimers during its stimulation by 2-5A. The protein dimers were observed to form only upon binding to 2-5A, as shown using gel filtration chromatography and chemical cross-linking and after centrifugation in glycerol gradients. A monoclonal antibody to 2-5A-dependent RNase was prepared and used to probe the subunit structure of the enzyme in the presence or absence of 2-5A. Using oligoadenylates of different length, structure, and 5'-phosphorylation states we determined that conversion of 2-5A-dependent RNase from its monomeric, inactive form to its homodimeric, active form required the presence of functional 2-5A. These results demonstrate that the catalytically active form of 2-5A-dependent RNase is a homodimer.

Evidence for a dimeric intermediate on the crystallization pathway of ribonuclease A.

Early steps in the crystallization process of pancreatic ribonuclease have been investigated by time-dependent fluorescence anisotropy, using a labeled protein as a fluorescent probe. Previous experiments have shown that steady-state fluorescence anisotropy is sensitive to protein-protein interactions and can be used to find new crystallization conditions. The present work describes an attempt, by means of time-resolved experiments, to detect and characterize species appearing in the early stages of the crystallization pathway. Fluorescence anisotropy decay was measured with synchrotron radiation as a light source under a variety of conditions where it is known that the solutions tend towards crystallization; the decay was analyzed by a maximum-entropy method that calculates a rotational correlation-time distribution. Fluorescence anisotropy originates in the Brownian rotatory motion of macromolecules and the values of the correlation times are related to the size and shape of different species present in the solution. In the presence of high salt concentrations, a bimodal distribution is always observed. Whereas a peak of protein monomer is still present, a second peak appears as a stable intermediate in the crystallization pathway. The correlation time of this new species varies between two and three times the correlation time of the monomer. The second peak is possibly the symmetrical dimer of the ribonuclease molecules commonly observed in all the high-salt crystal forms.

Crystal packing in six crystal forms of pancreatic ribonuclease

We compare the molecular packing of bovine pancreatic ribonuclease A (RNase A) in six crystal forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which bury 1570 Å 2 to 2790 Å 2 of the RNase surface. Regions of the protein surface involved in the six packings cover almost the whole RNase molecule. The abundance of polar interactions, about one per 200 Å 2, is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large interface covering 1800 Å 2 and eight to ten polar interactions. Its presence in the three salt-grown crystal forms suggests that it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms contain only small interfaces, implying a different mechanism of nucleation.

Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone.

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.

T cell recognition of bovine ribonuclease. Self/non-self discrimination at the level of binding to the I-Ak molecule.

Bovine RNase A specific T-cell hybridomas were generated to study the recognition of foreign Ag by T lymphocytes. One hybrid, TS12, was shown to recognize RNase in association with I-Ak. This hybridoma required bovine RNase to be processed before recognition. The immunogenic determinant on the RNase molecule recognized by TS12 was localized to the tryptic fragment RNase(40-61). All of the stimulatory ability of this determinant was shown to be contained within the synthetic 14mer RNase(43-56). When this segment of bovine RNase was compared with the self murine sequence, only one amino acid difference was found, a substitution of a proline residue at position 50 for a serine residue. This substitution completely abolishes binding to the I-Ak molecule, as shown by both functional and direct binding assays. This finding shows that self/non-self discrimination not only occurs at the level of the T cell, but also can be caused by an inability of the self peptide to associate with a class II molecule.

Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases.

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.

Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements.

The effects of proline and X-Pro peptide bond conformations on the fluorescence properties of tyrosine in peptides corresponding to parts of a proposed chain-folding initiation site in bovine pancreatic ribonuclease A are examined by time-resolved and steady-state fluorescence spectroscopy. In peptides with Tyr-Pro sequences, the conformational constraints of proline on a preceding residue result in significant fluorescence quenching for both trans and cis peptide bond conformations. Small peptides containing Pro-Tyr sequences, on the other hand, do not exhibit fluorescence quenching compared to Ac-Tyr-NHMe. Studies of fluorescence decay in the tryptic fragment of performic acid oxidized ribonuclease corresponding to residues 105-124 (i.e., O-T-16) demonstrate the presence of at least two environments of the single tyrosine chromophore (in the sequence Asn113-Pro114-Tyr115). In these two (ensemble-averaged) environments, tyrosine has shorter and longer lifetimes, respectively, than in Ac-Tyr-NHMe. The fluorescence heterogeneity in O-T-16 does not correlate with X-Pro cis/trans conformational heterogeneity that can be detected by nuclear magnetic resonance (NMR) spectroscopy. Instead, the fluorescence heterogeneity in O-T-16 arises from the presence of multiple conformations with the same X-Pro peptide bond conformations which interconvert rapidly on the 1H NMR time scale (tau much less than 1 ms) but are distinguishable on the fluorescence lifetime time scale (tau greater than or equal to 1 ns). From comparisons with the tyrosine fluorescence decay of smaller synthetic peptides, it is concluded that the long-lifetime tyrosine fluorescence component of O-T-16 arises from interactions involving residues outside the Asn113-Pro114-Tyr115-Val116-Pro117 sequence, which either stabilize particular local conformations in the vicinity of Tyr115 or act directly to protect Tyr115 from efficient fluorescence quenching. The short-lifetime component of O-T-16 is also observed for the pentapeptide Ac-Asn-Pro-Tyr-Val-Pro-NHMe. The data provide evidence for a nonrandom polypeptide conformation of O-T-16 under conditions of solvent pH and temperature at which the complete disulfide-intact ribonuclease molecule is fully folded. Implications of this work for the interpretation of fluorescence-detected unfolding experiments are discussed.

The mechanism of binding of a polynucleotide chain to pancreatic ribonuclease.

The crystalline complex of pancreatic ribonuclease (RNase) with oligomers of d(pA)4 has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.22 at 2.5 angstrom resolution. The asymmetric unit is a complex of one RNase molecule associated with four d(pA)4 oligomers. Although the DNA in this complex is segmented, and therefore shows some discontinuities, it nevertheless traces a continuous path 12 nucleotides in length that passes through the active site cleft of the enzyme and over the surface of the protein. The DNA makes a series of eight to nine electrostatic bonds between its phosphate groups and lysine and arginine residues on the protein, as well as specific chemical interactions at the active site. The path described by the sequence of nucleotides is likely to be that taken by an extended polynucleotide chain when it is bound by the enzyme.

Crystal structure of RNase A complexed with d(pA) 4

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA) 4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 Å resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA) 4 complex was calculated to 3.5 Å. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 Å resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 Å resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA) 4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA) 4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5′ nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5′ terminus by a salt link to lysine 98, passes near arginine 39 and then through a solvent region to terminate with its 3′ end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA) 4 is bound at its 5′ end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5′ phosphate of one tetramer and the 3′ bases of two other tetramers and this may help to stabilize the crystalline complex. If the sites of interaction between the protein and the d(pA) 4 fragments are mapped on the surface of the protein, they describe a nearly continuous path into and through the active site, across the surface of the enzyme and finally into the basic amino acid cluster on the opposite side of the protein. Such a virtual DNA strand could explain the observation that, when RNase binds to a long single strand of nucleic acid, it can cover or protect 11 to 12 bases along the polynycleotide chain. The path would also account for the observation that at least seven individual electrostatic interactions are involved in the binding of RNase to single-stranded nucleic acids.