Purification and characterization of Escherichia coli RNase T

RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.     

A novel cross-linked RNase A dimer with enhanced enzymatic properties

A new cross-linked ribonuclease A (RNase A) dimer composed of monomeric units covalently linked by a single amide bond between the side-chains of Lys(66) and Glu(9) is described. The dimer was prepared in the absence of water by incubating a lyophilized preparation of RNase, sealed under vacuum, in an oven at 85 degrees C. It was determined that the in vacuo procedure does not induce any significant conformational changes to the overall structure of RNase A, yet the amide cross-link has an increased acid lability, indicating that it is exposed and conformationally strained. Examination of X-ray crystallographic structures indicates that Lys(66) and Glu(9) are not close enough for the in vacuo dimer to adopt any of the known domain-swapped conformations. Therefore, the in vacuo RNase A dimer appears to be a novel dimeric structure. The in vacuo RNase A dimer also exhibits a twofold increase in activity over monomeric RNase A on a per monomer basis. This doubling of enzymatic activity was shown using dsRNA and ssRNA as substrates. In addition to this enhanced ability to degrade RNA, the dimer is not inhibited by the cellular ribonuclease inhibitor protein (cRI).     

Formation, structure, and dissociation of the ribonuclease S three-dimensional domain-swapped dimer

Post-translational events, such as proteolysis, are believed to play essential roles in amyloid formation in vivo. Ribonuclease A forms oligomers by the three-dimensional domain-swapping mechanism. Here, we demonstrate the ability of ribonuclease S, a proteolytically cleaved form of ribonuclease A, to oligomerize efficiently. This unexpected capacity has been investigated to study the effect of proteolysis on oligomerization and amyloid formation. The yield of the RNase S dimer was found to be significantly higher than that of RNase A dimers, which suggests that proteolysis can activate oligomerization via the three-dimensional domain-swapping mechanism. Characterization by chromatography, enzymatic assays, and NMR spectroscopy indicate that the structure of the RNase S dimer is similar to that of the RNase A C-dimer. The RNase S dimer dissociates much more readily than the RNase A C-dimer does. By measuring the dissociation rate as a function of temperature, the activation enthalpy and entropy for RNase S dimer dissociation were found to resemble those for the release of the small fragment (S-peptide) from monomeric RNase S. Excess S-peptide strongly slows RNase S dimer dissociation. These results strongly suggest that S-peptide release is the rate-limiting step of RNase S dimer dissociation.     

Degradation of double-stranded RNA by human pancreatic ribonuclease: crucial role of noncatalytic basic amino acid residues

Under physiological salt conditions double-stranded (ds) RNA is resistant to the action of most mammalian extracellular ribonucleases (RNases). However, some pancreatic-type RNases are able to degrade dsRNA under conditions in which the activity of bovine RNase A, the prototype of the RNase superfamily, is essentially undetectable. Human pancreatic ribonuclease (HP-RNase) is the most powerful enzyme to degrade dsRNA within the tetrapod RNase superfamily, being 500-fold more active than the orthologous bovine enzyme on this substrate. HP-RNase has basic amino acids at positions where RNase A shows instead neutral residues. We found by modeling that some of these basic charges are located on the periphery of the substrate binding site. To verify the role of these residues in the cleavage of dsRNA, we prepared four variants of HP-RNase: R4A, G38D, K102A, and the triple mutant R4A/G38D/K102A. The overall structure and active site conformation of the variants were not significantly affected by the amino acid substitutions, as deduced from CD spectra and activity on single-stranded RNA substrates. The kinetic parameters of the mutants with double-helical poly(A).poly(U) as a substrate were determined, as well as their helix-destabilizing action on a synthetic DNA substrate. The results obtained indicate that the potent activity of HP-RNase on dsRNA is related to the presence of noncatalytic basic residues which cooperatively contribute to the binding and destabilization of the double-helical RNA molecule. These data and the wide distribution of the enzyme in different organs and body fluids suggest that HP-RNase has evolved to perform both digestive and nondigestive physiological functions.     

The inside-out mechanism of Dicers from budding yeasts

The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function.     

Cytotoxicity of polyspermine-ribonuclease A and polyspermine-dimeric ribonuclease A

Polyspermine-ribonuclease A (PS-RNase A) and polyspermine-dimeric ribonuclease A (PS-dimeric RNase A) were prepared by cross-linking ribonuclease A or its covalently linked dimer to polyspermine (PS) using dimethyl suberimidate. The two RNase A derivatives were tested for a possible antitumor action. The in vitro and in vivo cytotoxic activity of PS-RNase A, although strong, is not higher than that known for free polyspermine. PS-dimeric RNase A, which was characterized by mass spectroscopy, titration of free amine groups, and enzymatic assays, proved instead to be a definitely more efficient antitumor agent, both in vitro and in vivo. This result could tentatively be explained in view of the importance of positive charges for ribonuclease activity, considering the higher basicity of PS-dimeric RNase A compared to that of PS-(monomeric)RNase A. It must be also taken into account that the dimeric RNase A moiety of PS-dimeric RNase A could evade the cytoplasmic ribonuclease inhibitor, which instead could trap the monomeric RNase A moiety of the other derivative. The two RNase A derivatives degrade poly(A).poly(U) under conditions where native RNase A is inactive. The results of this work demonstrate once again the importance of positive charges for the functions of mammalian pancreatic type ribonucleases in general, in particular for RNase A derivatives, and the potential therapeutic use of the ribonuclease A derivatives.     

Structural versatility of bovine ribonuclease A. Distinct conformers of trimeric and tetrameric aggregates of the enzyme.

Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII-A) from 40% acetic acid solutions leads to the formation of approximately 14 aggregated species that can be separated by ion-exchange chromatography. Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously [Gotte, G. & Libonati, M. (1998) Two different forms of aggregated dimers of ribonuclease A. Biochim. Biophys. Acta 1386, 106-112]. We also have possible evidence for the existence of two forms of pentameric RNase A. The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion-exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions. Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues. All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A).poly(U), viral double-stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species. On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A.     

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.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

Dimer formation by a "monomeric" protein

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S-peptide and S-protein) of the protein. The H12A and H119A variants have 10(5)-fold less ribonucleolytic activity than does the wild-type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65 degrees C results in a 10(3)-fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65 degrees C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer-dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37 degrees C. This value of Kd is only 20-fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of "monomeric" proteins could be more prevalent than is usually appreciated.     

Dimer dissociation and thermosensitivity kinetics of the Saccharomyces cerevisiae and human TATA binding proteins.

A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of the yeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted. We find that yeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t(1/2) approximately 20 min). Mild mutations in the crystallographic dimer interface accelerate the rate of dimer dissociation, whereas severe mutations prevent dimerization. In the presence of excess TATA DNA, which measures the entire active TBP population, dimer dissociation represents the rate-limiting step in DNA binding. These findings provide a biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulated gene expression in yeast [Jackson-Fisher, A. J., Chitikila, C., Mitra, M., and Pugh, B. F. (1999) Mol. Cell 3, 717-727]. In the presence of vast excesses of TBP over TATA DNA, which measures only a very small fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNA rapidly, which is consistent with a simultaneous binding and bending of the DNA. Under conditions where other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in the absence of TATA DNA, even at temperatures as low as 0 degrees C. Kinetic analyses of TBP instability in the absence of DNA at 30 degrees C revealed that even under fairly stabilizing solution conditions, TBP's DNA binding activity rapidly dissipated with t(1/2) values ranging from 6 to 26 min. TBP's stability appeared to vary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBP inactivation.     

Role for bovine viral diarrhea virus Erns glycoprotein in the control of activation of beta interferon by double-stranded RNA

Production of alpha/beta interferon in response to viral double-stranded RNA (dsRNA) produced during viral replication is a first line of defense against viral infections. Here we demonstrate that the Erns glycoprotein of the pestivirus bovine viral diarrhea virus can act as an inhibitor of dsRNA-induced responses of cells. This effect is seen whether Erns is constitutively expressed in cells or exogenously added to the culture medium. The Erns effect is specific to dsRNA since activation of NF-kappaB in cells infected with Semliki Forest virus or treated with tumor necrosis factor alpha was not affected. We also show that Erns contains a dsRNA-binding activity, and its RNase is active against dsRNA at a low pH. Both the dsRNA binding and RNase activities are required for the inhibition of dsRNA signaling, and we discuss here a model to account for these observations.     

Purification and characterization of a new ribonuclease from fruiting bodies of the oyster mushroom Pleurotus ostreatus.

A ribonuclease (RNase), possessing an N-terminal sequence disparate from those of ribonucleases from other mushrooms and previously isolated Pleuotus ostreatus RNases, was purified from the fruiting bodies of the edible mushroom Pleurotus ostreatus. The N-terminal sequence of Pleurotus ostreatus RNase did not manifest homology even to a previously reported RNase from the same mushroom. The ribonuclease was adsorbed on CM-Sepharose and Mono S. It exhibited a molecular mass of 12 kDa in both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease displayed an activity of 11490 U/mg on yeast tRNA. The highest ribonuclease activity was exhibited toward poly U, followed by poly A and poly C. No activity was shown toward poly G. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 240 nM.     

The major birch pollen allergen, Bet v 1, shows ribonuclease activity

The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units · mg^?1.     

Cytotoxicity of bovine seminal ribonuclease: monomer versus dimer

Bovine seminal ribonuclease (BS-RNase) is a homologue of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, BS-RNase has notable toxicity for human tumor cells. Wild-type BS-RNase is a homodimer linked by two intermolecular disulfide bonds. This quaternary structure endows BS-RNase with resistance to inhibition by the cytosolic ribonuclease inhibitor protein (RI), which binds tightly to RNase A and monomeric BS-RNase. Here, we report on the creation and analysis of monomeric variants of BS-RNase that evade RI but retain full enzymatic activity. The cytotoxic activity of these monomeric variants exceeds that of the wild-type dimer by up to 30-fold, indicating that the dimeric structure of BS-RNase is not required for cytotoxicity. Dimers of these monomeric variants are more cytotoxic than wild-type BS-RNase, suggesting that the cytotoxicity of the wild-type enzyme is limited by RI inhibition following dissociation of the dimer in the reducing environment of the cytosol. Finally, the cytotoxic activity of these dimers is less than that of the constituent monomers, indicating that their quaternary structure is a liability. These data provide new insight into structure-function relationships of BS-RNase. Moreover, BS-RNase monomers described herein are more toxic to human tumor cells than is any known variant or homologue of RNase A including Onconase, an amphibian homologue in phase III clinical trials for the treatment of unresectable malignant mesothelioma.     

Channels Formed by Amphotericin B Covalent Dimers Exhibit Rectification

Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.     

RNase 7 Contributes to the Cutaneous Defense against Enterococcus faecium

Background

Human skin is able to mount a fast response against invading microorganisms by the release of antimicrobial proteins such as the ribonuclease RNase 7. Because RNase 7 exhibits high activity against Enterococcus faecium the aim of this study was to further explore the role of RNase 7 in the cutaneous innate defense system against E. faecium.

Methodology/Principal Findings

Absolute quantification using real-time PCR and ELISA revealed that primary keratinocytes expressed high levels of RNase 7. Immunohistochemistry showed RNase 7 expression in all epidermal layers of the skin with an intensification in the upper more differentiated layers. Furthermore, RNase 7 was secreted by keratinocytes in vitro and in vivo in a site-dependent way. RNase 7 was still active against E. faecium at low pH (5.5) or high NaCl (150 mM) concentration and the bactericidal activity of RNase 7 against E. faecium required no ribonuclease activity as shown by recombinant RNase 7 lacking enzymatic activity. To further explore the role of RNase 7 in cutaneous defense against E. faecium, we investigated whether RNase 7 contributes to the E. faecium killing activity of skin extracts derived from stratum corneum. Treatment of the skin extract with an RNase 7 specific antibody, which neutralizes the antimicrobial activity of RNase 7, diminished its E. faecium killing activity.

Conclusions/Significance

Our data indicate that RNase 7 contributes to the E. faecium-killing activity of skin extracts and suggest an important role for RNase 7 in the protection of human skin against E. faecium colonization.     

Preparation of monoclonal antibodies against a poly(U), poly(C) specific ribonuclease prepared from the insect Ceratitis capitata.

A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.     

Kinetic mechanism for formation of the active,dimeric UvrD helicase-DNA complex

Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.