Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([³H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-γ (IFN-γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-γ's ability to control cell growth and induce an antiviral state.     

A new pyrimidine-specific ribonuclease from bovine seminal plasma that is active on both single and double-stranded polyribonucleotides and that can distinguish between Mg2+-containing and Mg2+-depleted naturally occurring RNAs

The purification to homogeneity of a new ribonuclease, named RNAase SPL, from bovine seminal plasma is described. This nuclease, like the bovine pancreatic RNAase A, is pyrimidine specific. Its activity on single-stranded synthetic polyribonucleotides such as poly(rU) is significantly higher than that of RNAase A. However, unlike RNAase A, RNAase SPL is highly active on a double-stranded RNA such as poly[r(A · U)], and shows extremely limited activity on naturally occurring RNAs, such as Escherichia coli RNA, prepared with Mg²⁺ present throughout the isolation procedure. Under conditions of limiting hydrolysis in which RNAase A degrades 60 to 90% of total E. coli RNA to acid-soluble material and the remaining to material having a molecular weight lower than that of transfer RNA, RNAase SPL does not yield any acid-soluble products: it does not appear to degrade tRNA or 5 S RNA, and causes only a small number of nicks in the remaining RNAs to yield a limiting digest containing products with molecular weights ranging between 10,000 and 150,000. Absence of Mg²⁺ during the isolation procedure, or heat denaturation of the RNA makes it as susceptible to RNAase SPL as it is to RNAase A.The above and other related observations reported here support the view that there are Mg²⁺-dependent structural features, besides single and doublestrandedness, in naturally occurring RNAs, that can be distinguished by using the two nucleases RNAase SPL and RNAase A.     

The eight human “canonical” ribonucleases: Molecular diversity, catalytic properties, and special biological actions of the enzyme proteins

Human ribonucleases (RNases) are members of a large superfamily of rapidly evolving homologous proteins. Upon completion of the human genome, eight catalytically active RNases (numbered 1-8) were identified. These structurally distinct RNases, characterized by their various catalytic differences on different RNA substrates, constitute a gene family that appears to be the sole vertebrate-specific enzyme family. Apart from digestion of dietary RNA, a wide variety of biological actions, including neurotoxicity, angiogenesis, immunosuppressivity, and anti-pathogen activity, have been recently reported for almost all members of the family. Recent evolutionary studies suggest that RNases started off in vertebrates as host defence or angiogenic proteins.     

Structural and functional relationships of natural and artificial dimeric bovine ribonucleases: New scaffolds for potential antitumor drugs

Protein aggregation via 3D domain swapping is a complex mechanism which can lead to the acquisition of new biological, benign or also malignant functions, such as amyloid deposits. In this context, RNase A represents a fascinating model system, since by dislocating different polypeptide chain regions, it forms many diverse oligomers. No other protein displays such a large number of different quaternary structures. Here we report a comparative structural analysis between natural and artificial RNase A dimers and bovine seminal ribonuclease, a natively dimeric RNase with antitumor activity, with the aim to design RNase A derivatives with improved pharmacological potential.     

Studies of the RNA degradosome-organizing domain of the Escherichia coli ribonuclease RNase E

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10-40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein-RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.     

Presence and distribution of double-stranded RNA elements in the white root rot fungus Rosellinia necatrix

Double-stranded (ds)RNA of various types was detected in 65 (21.8%) of 298 isolates from vegetative hyphae of Rosellinia necatrix by electrophoresis, but dsRNA was not detected from 39 ascosporic isolates. There were 45 distinct dsRNA profiles in the 65 isolates: they varied in the number of electrophoretic bands from 1 to 12 and in size from less than 1000 bp to more than 10 kbp. Each dsRNA profile was unique to each locality. dsRNAs having the same profiles were restricted to isolates of the same mycelial compatibility groups (MCG) from the same trees, with an exception where different profiles were detected in different isolates of the same MCGs. Received: May 7, 2001 / Accepted: September 5, 2001     

Aspartic acid-121 functions at the active site of bovine pancreatic ribonuclease

6-Phosphofructo-2-kinase (PFK2) is activated by a cAMP-dependent protein kinase, and inactivated by phosphatase, indicating the interconversion of PFK2. Inorganic phosphate also activates PFK2, and the optimum pH for the PFK2 activity varies with the concentration of phosphate. Phosphate also enhances the inactivation of PFK2 by citrate, suggesting that phosphate acts as a regulator of PFK2.     

Solution structures of the double-stranded RNA-binding domains from RNA helicase A

RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA.     

Different mechanisms of action of poly(ethylene glycol) and arginine on thermal inactivation of lysozyme and ribonuclease A

Proteins tend to undergo irreversible inactivation through several chemical modifications, which is a serious problem in various fields. We have recently found that arginine (Arg) suppresses heat‐induced deamidation and β‐elimination, resulting in the suppression of thermal inactivation of hen egg white lysozyme and bovine pancreas ribonuclease A. Here, we report that poly(ethylene glycol) (PEG) with molecular weight 1,000 acts as a thermoinactivation suppressor for both proteins, especially at higher protein concentrations, while Arg was not effective at higher protein concentrations. This difference suggests that PEG, but not Arg, effectively inhibited intermolecular disulfide exchange among thermally denatured proteins. Investigation of the effects of various polymers including PEG with different molecular weight, poly(vinylpyrolidone) (PVP), and poly(vinyl alchol) on thermoinactivation of proteins, circular dichroism, solution viscosity, and the solubility of reduced and S‐carboxy‐methylated lysozyme indicated that amphiphilic PEG and PVP inhibit intermolecular collision of thermally denatured proteins by preferential interaction with thermally denatured proteins, resulting in the inhibition of intermolecular disulfide exchange. These findings regarding the different mechanisms of the effects of amphiphilic polymers––PEG and PVP––and Arg would expand the capabilities of methods to improve the chemical stability of proteins in solution. Biotechnol. Bioeng. 2012; 109: 2543–2552. © 2012 Wiley Periodicals, Inc.     

Glycine 38 is crucial for the ribonucleolytic activity of human pancreatic ribonuclease on double-stranded RNA

Evidence is now in favor of protein-facilitated mechanisms for the intestinal cholesterol absorption. Here we report that the unesterified cholesterol uptake by rat jejunal brush border membrane vesicles (BBMVs) is efficient, saturable, and protein-mediated. The human apolipoproteins biliary anionic peptide factor (APF) and A-I (apoA-I) up-regulate micellar cholesterol uptake in a dose-dependent manner, but for all tested concentrations (0.1-20 microM), the lipid-free APF was more efficient than apoA-I. This uptake stimulation was suppressed after addition of Pabs directed to the external lipid-binding domain of the CLA-1/SR-BI and reduced by Pabs directed to the external loop of CD36. Thus, CLA-1/SR-BI and to a lesser extent CD36 are involved in the regulation of intestinal cholesterol uptake. APF, the main protein bound to biliary lipids, is likely one of their physiological effectors. As APF is an unesterified cholesterol carrier, it could facilitate the intestinal absorption of biliary cholesterol.     

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.     

Interactions of the cytotoxic RNase A dimers with the cytosolic ribonuclease inhibitor

Ribonuclease A (RNase A) dimers have been recently found to be endowed with some of the special, i.e., non-catalytic biological activities of RNases, such as antitumor and aspermatogenic activities. These activities have been so far attributed to RNases which can escape the neutralizing action of the cytosolic RNase inhibitor (cRI). However, when the interactions of the two cytotoxic RNase A dimers with cRI were investigated in a quantitative fashion and at the molecular level, the dimers were found to bind cRI with high affinity and to form tight complexes.     

Mutations in domain a' of protein disulfide isomerase affect the folding pathway of bovine pancreatic ribonuclease A

Protein disulfide isomerase (PDI, EC 5.3.4.1), an enzyme and chaperone, catalyses disulfide bond formation and rearrangements in protein folding. It is also a subunit in two proteins, the enzyme collagen prolyl 4-hydroxylase and the microsomal triglyceride transfer protein. It consists of two catalytically active domains, a and a', and two inactive ones, b and b', all four domains having the thioredoxin fold. Domain b' contains the primary peptide binding site, but a' is also critical for several of the major PDI functions. Mass spectrometry was used here to follow the folding pathway of bovine pancreatic ribonuclease A (RNase A) in the presence of three PDI mutants, F449R, Delta455-457, and abb', and the individual domains a and a'. The first two mutants contained alterations in the last alpha helix of domain a', while the third lacked the entire domain a'. All mutants produced genuine, correctly folded RNase A, but the appearance rate of 50% of the product, as compared to wild-type PDI, was reduced 2.5-fold in the case of PDI Delta455-457, 7.5-fold to eightfold in the cases of PDI F449R and PDI abb', and over 15-fold in the cases of the individual domains a and a'. In addition, PDI F449R and PDI abb' affected the distribution of folding intermediates. Domains a and a' catalyzed the early steps in the folding but no disulfide rearrangements, and therefore the rate observed in the presence of these individual domains was similar to that of the spontaneous process.     

Three single nucleotide polymorphisms leading to non-synonymous amino acid substitution in the human ribonuclease 2 and angiogenin genes exhibit markedly less genetic heterogeneity in six populations

Angiogenin and ribonuclease 2 (RNase 2) are members of the human RNase superfamily. Although three potential single nucleotide polymorphisms (SNPs) in these genes, which could give rise to an amino acid substitution in the protein, have been identified, relevant population data are not available, and accordingly they have not been applied to clinical-genetic analysis. For this purpose, a novel genotyping method for each SNP using the mismatched PCR-restriction fragment length polymorphism technique has been developed. Using this method, the genotype distribution of each SNP was investigated in six populations: Japanese (n = 167), Korean (n = 90), Mongolian (n = 92), Ovambos (n = 86), Turkish (n = 87), and German (n = 70). In all the populations, only one genotype was found in each SNP. Irrespective of differences in ethnic groups, the angiogenin and RNase 2 genes appear to exhibit markedly less genetic heterogeneity with regard to these SNPs.     

Experimental Evidence That GNA and TNA Were Not Sequential Polymers in the Prebiotic Evolution of RNA

Systematic investigation into the chemical etiology of ribose has led to the discovery of glycerol nucleic acid (GNA) and threose nucleic acid (TNA) as possible progenitor candidates of RNA in the origins of life. Coupled with their chemical simplicity, polymers for both systems are capable of forming stable Watson-Crick antiparallel duplex structures with themselves and RNA, thereby providing a mechanism for the transfer of genetic information between successive genetic systems. Investigation into whether both polymers arose independently or descended from a common evolutionary pathway would provide additional constraints on models that describe the emergence of a hypothetical RNA world. Here we show by thermal denaturation that complementary GNA and TNA mixed sequence polymers are unable, even after prolonged incubation times, to adopt stable helical structures by intersystem cross-pairing. This experimental observation suggests that GNA and TNA, whose structures derive from one another, were not consecutive polymers in the same evolutionary pathway to RNA. Reviewing Editor: Dr. Niles Lehman     

An S-like ribonuclease gene is used to generate a trap-leaf enzyme in the carnivorous plant Drosera adelae

Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack.     

Elucidation of the ribonuclease A aggregation process mediated by 3D domain swapping: a computational approach reveals possible new multimeric structures

By lyophilization from 40% acetic acid solutions, bovine pancreatic ribonuclease A forms several three-dimensional (3D) domain-swapped oligomers: dimers, trimers, tetramers, pentamers, hexamers, and traces of high-order oligomers, purifiable by cation-exchange chromatography. Each oligomeric species consists of at least two conformers displaying different basicity density, and/or exposure of positive charges. The structures of the two dimers and one trimer have been solved. Plausible models have been proposed for a second RNase A trimer and four tetramers, but not all the models are certainly assignable to the tetramers purified. Further studies have also been made on the pentameric and hexameric species, again without reaching structurally clear-cut results. This work is focused on the detailed modeling of the tetrameric RNase A species, using four different approaches to possibly clarify unknown structural aspects. The results obtained do not confirm the validity of one tetrameric model previously proposed, but allow the proposal of a novel tetrameric structure displaying new interfaces that are absent in the other known conformers. New details concerning other tetrameric structures are also described. RNase A multimers larger than tetramers, i.e., pentamers, hexamers, octamers, nonamers, up to dodecamers, are also modeled, with the proposal of novel domain-swapped structures, and the confirmation of what had previously been inferred. Finally, the propensity of RNase A to possibly form high-order supramolecular multimers is analyzed starting from the large number of domain-swapped RNase A conformers modeled.     

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.