Aminoglycosides suppress tRNA processing in human epidermal keratinocytes in vitro.

The ever-growing resistance of pathogens to antibiotics and the lack of potent antibacterial drugs constitute major problems in the treatment of infectious diseases. Thus, the better understanding of the mode of action of antibiotics at the molecular level is of essential importance. Accumulating evidence points towards RNA as being a crucial target of antibacterial and antiviral drugs. Interestingly, aminoglycosides, one of the most important families of antibiotics, apart from their inhibitory effect on ribosome function, reportedly interfere with various RNA molecules and in vitro suppress the proliferation of human keratinocytes. In this study we investigated the effect of the aminoglycosides neomycin B, paromomycin, tobramycin and gentamycin on ribonuclease P activity from normal human epidermal keratinocytes. All aminoglycosides tested revealed a dose-dependent inhibition of tRNA maturation, which was reduced by increasing Mg(2+) ion concentrations, indicating competition of the cationic aminoglycosides with magnesium ions required for catalysis. Our in vitro findings suggest that the inhibitory effects of aminoglycosides on tRNA processing may be implicated in the mechanisms of their antiproliferative action on human epidermal keratinocytes.

Calcipotriol plus short-contact dithranol: a novel topical combination therapy for chronic plaque psoriasis.

The purpose of this double-blind randomised parallel-group study was to compare the efficacy and safety of short-contact treatment with dithranol ointment (2%) with its combination with calcipotriol ointment (50 microg/g) in 2 groups of in-patients with chronic plaque psoriasis. The patients of the first group (n = 23) topically applied dithranol once daily for 30 min and the vehicle of calcipotriol twice daily. The patients of the second group (n = 23) used a single topical application of dithranol for 30 min daily and additionally applied calcipotriol twice daily. The extent and the severity of psoriasis were assessed by means of psoriasis area and severity index score (PASI score) before the onset of the 6-week therapy and weekly thereafter. The difference between the two groups with regard to the mean PASI score became statistically significant already after the first week of treatment and remained so until the end of the trial. No significant differences were observed between the two groups with respect to the cutaneous adverse events. These findings indicate that the addition of calcipotriol ointment to short-contact dithranol markedly augments the therapeutic efficacy of the latter in chronic plaque psoriasis and impressively accelerates the response of psoriatic plaques to this well-tolerated regimen.

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Inhibitory effects of arotinoids on tRNA biogenesis.

The effects of five arotinoids (Ro 13-7410, Ro 15-0778, Ro 15-1570, Ro 13-6298, Ro 40-8757) on ribonuclease P (RNase P) activity were studied in a cell-free system derived from Dictyostelium discoideum. RNase P is a ribonucleoprotein that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. Kinetic analysis showed that these compounds behave as classical competitive inhibitors with Ki values 4.35, 3.6, 2.8 and 0.045 mM for Ro 13-6298, Ro 15-1570, Ro 15-0778 and Ro 13-7410, respectively. Ro 13-7410 was 62, 80 and 97 times more potent in inhibiting the enzyme activity as compared to Ro 15-0778, Ro 15-1570 and Ro 13-6298, respectively, whereas Ro 40-8757 showed no effect on RNase P activity. These results project the significance of the acidic polar terminus in the arotinoid molecule binding to the enzyme. The kinetics of inhibition reflects allosteric interactions of arotinoids with D. discoideum RNase P. Moreover, our findings indicate that the inhibitory effects of arotinoids on tRNA biogenesis can be mediated through mechanisms not involving the retinoid nuclear receptors.

Effect of peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum. Effect of antibiotics on RNase P.

The effect of several peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum was investigated. Among the inhibitors tested puromycin, amicetin and blasticidin S revealed a dose-dependent inhibition of tRNA maturation. Blasticidin S and amicetin do not compete with puromycin for the same site on the enzyme, suggesting the existence of distinct antibiotic binding sites on D. discoideum RNase P. Inhibition experiments further indicate that binding sites for blasticidin S and amicetin overlap.

Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies.

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non-competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg(2+) ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg(2+) for common binding sites on RNase P holoenzyme.

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Additive inhibitory effect of calcipotriol and anthralin on ribonuclease P activity.

The effects of two antipsoriatic compounds, calcipotriol and anthralin, separately or in combination on ribonuclease P (RNase P), were investigated using a cell-free system from the slime mold Dictyostelium discoideum. RNase P is an ubiquitous and essential enzyme which endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. The substrate for RNase P assays was an in vitro (32)P-labeled transcript of the Schizosaccharomyces pombe tRNA(Ser) gene supS1. Enzyme assays were carried out at 37 degrees in 20 microL 50 mM Tris-HCL 7.6 buffer, containing 10 mM NH(4)Cl, 5 mM MgCl(2), and 10% isopropanol. Calcipotriol or anthralin alone exerted a dose-dependent inhibitory effect on RNase P activity, with the former being more active than the latter in this respect. Simultaneous exposure of the enzyme to both drugs resulted in an enhancement of RNase P inhibition, which was additive. Considering the lack of structural similarities between the substrate (precursor tRNA) of RNase P and the tested drugs, it seems reasonable to suggest that their effects may be due to binding to allosteric inhibition sites of the enzyme. Although our in vitro findings cannot be directly extrapolated to the in vivo human condition, they do suggest that the inhibitory effects of calcipotriol and anthralin on tRNA biogenesis may be implicated in the mechanisms of their antipsoriatic action. Moreover, the additive inhibitory effect of these compounds on RNase P activity provides an experimental basis for their possible combined therapeutic application in the management of psoriasis.

Dose-dependent inhibition of ribonuclease P activity by anthralin.

The effect of five different anthralin concentrations on tRNA biogenesis was investigated employing the ribonuclease P (RNase P) of the slime mold Dictyostelium discoideum as an in vitro cell-free experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. Anthralin revealed a dose-dependent inhibition of RNase P activity indicating that this compound may have a direct effect on tRNA biogenesis. Taking into account that anthralin has no structural similarities to the substrate (pre-tRNA) of RNase P, it seems reasonable to suggest that this compound may bind to allosteric inhibition sites of the enzyme.

Modulation of ribonuclease P activity by calcipotriol.

The effects of cholesterol, 7-dehydrocholesterol, vitamin D3 and several synthetic vitamin D3 analogs on ribonuclease P (RNase P) were investigated using a cell-free system from the slime mold Dictyostelium discoideum. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. Among the compounds tested, only calcipotriol was capable of affecting RNase P activity, and revealed a bimodal action at the kinetic phase of the reaction. Depending on the concentration of the drug, both activation and inhibition of tRNA maturation were observed, indicating that calcipotriol may have a direct effect on tRNA biogenesis, possibly associated with the presence of a highly reactive small ring on the side chain of its molecule.

Bimodal action of alkaline earth cations on Dictyostelium discoideum ribonuclease P activity.

The ribonucleoprotein ribonuclease P (RNase P) cleaves all tRNA precursors endonucleolitically to produce the mature 5'-end. Dictyostelium discoideum RNase P displays an absolute requirement for Mg2+. Only the alkaline earth cations Ca2+, Sr2+, and Ba2+, under appropriate conditions can substitute to some extent for Mg2+. The transition metals Mn2+, Co2+, Ni2+, and Cd2+ are efficient inhibitors of the enzyme activity. Ca2+, Sr2+ and Ba2+, in the presence of Mg2+, exhibit a bimodal action at the kinetic phase of the reaction. Kinetic analysis of the activation phase revealed that Ca2+, Sr2+, or Ba2+ attached on a specific site of RNase P act as nonessential-noncompetitive activators. Further additions of Ca2+, Sr2+, or Ba2+ cause noncompetitive inhibition on the RNase P reaction, indicating that RNase P possesses a second binding site responsible for the inhibitory effect of Ca2+, Sr2+, and Ba2+. Both activator and inhibitory sites can be occupied by Ca2+, Sr2+, or Ba2+ at the same time.

Inhibition of ribonuclease P activity by retinoids.

The effect of two naturally occurring (retinol and all-trans retinoic acid) and two synthetic (isotretinoin and acitretin) analogs of vitamin A (retinoids) on tRNA biogenesis was investigated employing the RNase P of Dictyostelium discoideum as an in vitro experimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5' end. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classical competitive inhibitors. The Ki values determined were 1475 microM for retinol, 15 microM for all-trans retinoic acid, 20 microM for isotretinoin, and 8.0 microM for acitretin. On the basis of these values acitretin is a 184, 2.5, and 1.9 times more potent inhibitor, as compared with retinol, isotretinoin, and all-trans retinoic acid, respectively. Taking into account that retinoids share no structural similarities to precursor tRNA, it is suggested that their kinetic behavior reflects allosteric interactions of these compounds with hydrophobic site(s) of D. discoideum RNase P.

Determination of eukaryotic peptidyltransferase activity by pseudo-first-order kinetic analysis.

We have developed an in vitro system for the determination of peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA-poly(U)-80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate (k3) constant is identical to the catalytic rate constant (kcat) of peptidyltransferase. This k3 of peptidyltransferase is equal to 2.9 min-1 at 37 degrees C. Moreover, the ratio k3/ Ks, which is an accurate measure of peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic peptidyltransferase.

Partial purification and characterization of RNase P from Dictyostelium discoideum.

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.

The RNase P of Dictyostelium discoideum.

Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis. It catalyses the endonucleolytic cleavage of nearly all tRNA precursors to produce 5'-end matured tRNA. RNase P activity has been found in all organisms examined, from bacteria to mammals. Eubacterial RNase RNA is the only known RNA enzyme which functions in trans in nature. Similar behaviour has not been demonstrated in RNase P enzymes examined from archaebacteria or eukaryotes. Characterisation of RNase P enzymes from more diverse eukaryotic species, including the slime mold Dictyostelium discoideum, is useful for comparative analysis of the structure and function of eukaryotic RNase P.

Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions.

At 6 mM Mg2+, submillimolar concentrations of spermine affect the end-point as well as the kinetic phase of puromycin reaction in a cell-free system from Escherichia coli. When the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the absence of ribosomal wash (FWR fraction), the final degree of AcPhe-puromycin synthesis is raised from 12% to 60%, as the concentration of spermine increases from zero to 200 microM. However, spermine displays partial noncompetitive inhibition at the kinetic phase of the reaction. The inhibitory effect of spermine is related with its binding to AcPhe-tRNA. When complex C is formed in the presence of FWR fraction, spermine slightly affects the final degree of puromycin synthesis is markedly stimulated by the addition of relatively low concentrations of spermine. Kinetic analysis of the activation phase revealed that spermine attached on a specific site of complex C, acts as a nonessential, partial noncompetitive activator. The stimulatory effect of spermine seems to be due to its interaction with ribosomes. Further additions of spermine cause partial noncompetitive inhibition on the puromycin reaction. This result suggests that complex C possesses a second binding site, responsible for the inhibitory effect of spermine. Both activator and inhibitor sites can be occupied by spermine at the same time.

Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms.

The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface.

Aminoacyl analogs of chloramphenicol: examination of the kinetics of inhibition of peptide bond formation.

Two aminoacyl analogs and one peptidyl analog of chloramphenicol (1, Cl2CHCO-CA) were prepared. These are 2 (L-Phe-CA), 3 (Gly-CA), and 4 (L-Phe-Gly-CA). The kinetics of inhibition of peptide bond formation by these analogs were examined in a cell-free system which was derived from E. coli and used previously for the study of 1 (Drainas; et al. Eur. J. Biochem. 1987, 164, 53-58). In the absence of inhibitor, the reaction follows first-order kinetics for the entire course of the reaction. In the presence of the analog the reaction gives biphasic log-time plots. The kinetic information pertaining to the initial slope of the plot is analyzed (initial-slope analysis). This information differentiates the analogs from the parent compound 1. The parent compound 1 gives complex inhibition kinetics; increasing the concentration of 1 changes the inhibition from competitive to mixed noncompetitive (Drainas; et al. Eur. J. Biochem. 1987, 164, 53-58). In contrast, the analogs give competitive kinetics even at high concentrations of the inhibitor. The following Ki values have been determined: 18.0 microM for 2, 5.5 microM for 3, 1.5 microM for 4. If we were to assume that compounds 2, 3 and 4 behave as classical competitive inhibitors, we could say that 4 is 12 times more potent than 3 and 4 times more potent than 2. On this assumption we could also compare 1 with 4 and see that 4 is 2 times weaker than 1. It is suggested that as compared with 1, the two aminoacyl analogs and the dipeptidyl analog have increased structural similarity to the 3'-terminus of aminoacyl-tRNA or of peptidyl-tRNA and that this similarity results in a more pronounced competitive inhibition.

Identification of a 100-kDa protein associated with nuclear ribonuclease P activity in Schizosaccharomyces pombe.

Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent homogeneity. A purification of 23,000-fold was achieved by four fractionation steps with DEAE-cellulose chromatography, phosphocellulose chromatography, glycerol-gradient fractionation and finally tRNA-affinity chromatography. A 100-kDa protein was present in the most pure preparations in amounts approximately stoichiometric with the previously identified RNA components of the enzyme, K1-RNA and K2-RNA [Krupp, G., Cherayil, B., Frendeway, D., Nishikawa, S. & Söll, D. (1986) EMBO J. 5, 1697-1703]. A cross-linking experiment utilizing a 4-thiouridine-substituted precursor tRNA demonstrated that the 100-kDa protein interacts with the ribonuclease P substrate in a specific fashion. We therefore conclude that the protein component of S. pombe ribonuclease P is a 100-kDa protein.