Cytotoxic potential of ribonuclease and ribonuclease hybrid proteins

Pancreatic RNase injected into Xenopus oocytes abolishes protein synthesis at concentrations comparable to the toxin ricin yet has no effect on oocyte protein synthesis when added to the extracellular medium. Therefore RNase behaves like a potent toxin when directed into a cell. To explore the cytotoxic potential of RNase toward mammalian cells, bovine pancreatic ribonuclease A was coupled via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M whereas greater than 10(-5) M native RNase was required to inhibit protein synthesis. Cytotoxicity requires both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Compounds that interfere with transferrin receptor cycling and compartmentalization such as ammonium chloride decreased the cytotoxicity of transferrin-RNase. After a dose-dependent lag period inactivation of protein synthesis by transferrin-RNase followed a first-order decay constant. In a clonogenic assay that measures the extent of cell death 1 x 10(-6) M transferrin-RNase killed at least 4 logs or 99.99% of the cells whereas 70 x 10(-6) M RNase was nontoxic. These results show that RNase coupled to a ligand can be cytotoxic. Human ribonucleases coupled to antibodies also may exhibit receptor-mediated toxicities providing a new approach to selective cell killing possibly with less systemic toxicity and importantly less immunogenicity than the currently employed ligand-toxin conjugates.     

Production of an extracellular ribonuclease by Pseudomonas maltophilia.

As part of a screening program for pseudomonad enzymes having an industrial interest, we selected ribonuclease (RNase) producing strains. Of the 150 pseudomonads screened, 6 were found to produce an extracellular RNase activity when grown on solid medium. In broth culture, the RNase activity from these six species remained bound to the cells unless gelatin was added to the medium. Gelatin was essential for the release of RNase in the broth culture, but the pH of the medium, addition of potential inducers such as nucleic acids, or addition of cations did not affect this release. However, gelatin did not appear to induce the synthesis of the enzyme. Strain B-88, identified as Pseudomonas maltophilia, was selected for further study of the enzyme. The extracellular RNase isolated from B-88 broth cultures could be separated in two fractions on the basis of the molecular weight by the ultrafiltration technique. The low molecular weight fraction reacts optimally at temperatures between 55 and 60 degrees C and optimal pH values varying from 7.4 to 9.5. At neutral or alkaline pH, the enzyme was stable at temperatures below 37 degrees C but was inactivated at 55 degrees C. The RNase was inhibited by mercury and cobalt and stimulated by magnesium.     

Location of RNase and RNase inhibitor on free cytoplasmic mRNA-protein particles from human placenta

Free cytoplasmic mRNPs were isolated from human placenta. An activity of RNase was associated with these particles but was mostly inhibited by a labile protein inhibitor. Both RNase and RNase inhibitor were extractable from mRNPs by 0.5 M KCl. The nature of the association of the RNase-RNase inhibitor complex with mRNPs makes it suitable as a putative system for control of expression and turnover of mRNPs and therefore of protein synthesis.Abbreviations used RNase ribonuclease - mRNPs messenger ribonucleoprotein particles - pHMB p-hydroxymercuribenzoate     

Processing and secretion of the Yarrowia lipolytica RNase.

Secretion of the extracellular RNase from the yeast Yarrowia lipolytica was studied in pulse-chase and immunoprecipitation experiments. A polypeptide of 45,000 daltons was immunoprecipitated from [35S]methionine-labeled cell extracts and supernatant medium by rabbit anti-RNase antiserum. The RNase was secreted rapidly; the time between synthesis and appearance in the extracellular medium was about 5 min. In pulse-chase experiments, about 50% of the RNase was still cell associated 30 min after labeling. A polypeptide of 73,000 daltons whose immunoprecipitation was blocked by an excess of purified RNase was also detected. It broke down to a polypeptide with the same mobility and same peptide map as the mature RNase. Peptide maps of the undegraded 73-kilodalton polypeptide and the intracellular mature RNase contained several peptides of identical mobility. Immunoprecipitates from cells labeled in the presence of tunicamycin contained 66- and 45-kilodalton polypeptides. Endoglycosidase H treatment of the 73-kilodalton polypeptide converted it to a 66-kilodalton form, but did not change the apparent molecular weight of the mature form of the RNase. Labeling kinetics from pulse-chase experiments did not clearly support a precursor-product relationship between the 73-kilodalton polypeptide and the intracellular 45-kilodalton form of the RNase, and other relationships between the two polypeptides are possible.     

Effect of the RNAase from Bacillus intermedius on growth and physiological characteristics of Escherichia coli]

The effect of the RNase from Bacillus intermedius on the growth of Escherichia coli was investigated. RNase added to growth medium enhanced the synthesis of DNA, RNA, and protein and stimulated cell division; the degree of stimulation depended on the enzyme concentration. A necessary condition for stimulation was the adsorption of the enzyme on the cell surface and its interaction with the cytoplasmic membrane, as demonstrated immunocytochemically. The adsorption of the enzyme was accompanied by a 43% decrease in the surface charge density. Other effects of RNase involved a 25% increase in the growth rate, a 38% biomass gain, and generation time shortening by 10 min. The stimulation of bacterial growth correlated with the stimulation of cellular respiration rate.     

Interaction of ribonuclease H from Drosophila melanogaster embryos with DNA polymerase-primase

An RNase H was purified 2,500-fold to near homogeneity from early embryos of Drosophila melanogaster. The purified enzyme has an approximate molecular weight of 180,000 and appears to consist of two 49,000- and two 39,000-dalton polypeptides. The enzyme specifically hydrolyzes RNA.DNA hybrids and releases oligoribonucleotides ranging in size from 2-9 residues. The RNase H can also remove RNA primers that are synthesized and subsequently elongated by the Drosophila polymerase-primase. Preincubation of the RNase H from D. melanogaster embryos with the homologous DNA polymerase-primase results in an increased rate of DNA synthesis. The DNA chains synthesized under these conditions are shorter than those synthesized in the absence of the RNase H, and the rate of primer synthesis is increased significantly. These findings suggest that the RNase H forms a complex with the polymerase-primase, increasing its recycling capacity and thereby increasing the frequency of chain initiation.     

Stigmatic RNase in calamondin (Citrus reticulata var. austera x Fortunella sp.)

In calamondin. which is self-compatible, ribonuclease (RNase) activity was found in the stigmatic diffusate. Tissue print experiments using calamondin styles demonstrated that most of the RNase is localized in the stigma. Stigmatic RNase activity was monitored at different developmental stages of the flower and was found to peak at anthesis. An SDS-PAGE-zymogram indicated the molecular mass of this RNase to be 24 kDa. In vitro germination of calamondin pollen showed a higher percentage of germination in the presence of diffusate from one stigma than in the control. However, diffusates from 3.5 and 7 stigmata per 100 μl aliquots of growth medium, exhibiting 15. 25 and 35 units ml⁻¹ RNase activity, respectively, had successively stronger inhibition effects on the percentage of germination. Diffusate from 7 stigmata inhibited pollen tube elongation, as well as pollen germination. Both the 24-kDa Stigmatic RNase and RNase TI significantly inhibited pollen germination and pollen tube elongation. In pollen tubes treated with either the 24-kDa stigmatic RNase or RNase TI, considerable deposition of callose was observed, as compared to the control which had only a thin callosic cell wall.     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.     

The role of the promoter and leader sequences of extracellular ribonuclease from Bacillus amyloliquefaciens in regulation of enzyme biosynthesis

It is shown that in the medium rich with inorganic phosphate there is a stimulation of biosynthesis of ribonuclease from B. amyloliquefaciens (barnase) by actinomycin D, while biosynthesis of ribonucleases from B. intermedius (binase) and B. pumilus (KNase Bpu) in these conditions was suppressed. Features of biosynthesis of binase and RNase Bpu, directed by the barnase promoter, and also expression of chimeric gene of RNase Bpu with leader peptide of barnase were investigated. It was established that stimulation of synthesis of extracellular ribonuclease from B. amyloliquefaciens in the presence of actinomycin D was defined by structure of leader sequences.     

The effect of nicotinamide on unscheduled DNA synthesis in cultured hepatocytes

The synthesis of $\text{E. coli}$ proteins was examined, by two-dimensional O'Farrell gels, in mutant strains defective in all possible combinations of the RNA processing enzymes RNase III, RNase E and RNase P. We found that the synthesis of most proteins was unaffected; however, the synthesis of a significant number of proteins, 21 out of 80 tested, was drastically reduced in the strain defective in all three enzymes. It appears that the two enzymes RNase III and RNase E are responsible for most of these changes.     

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.     

Amplified Rnase H Activity in Escherichia coli B/R Increases Sensitivity to Ultraviolet Radiation

Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.     

Both temperature and medium composition regulate RNase E processing efficiency of the rpsO mRNA coding for ribosomal protein S15 of Escherichia coli

Cleavage by RNase E is believed to be the rate-limiting step in the degradation of many RNAs. These cleavages are modulated by 5' end-phosphorylation, folding and translation of the mRNA in question. Here, we present data suggesting that these cleavages are also regulated by environmental conditions. We report that rpsO mRNA, 15 minutes after a shift to 44 degrees C, is stabilized in cells grown in minimal medium. This stabilization is correlated with a reduction in the efficiency of the RNase E cleavage which initiates its decay. We also observe the appearance of RNA fragments previously detected following RNase E inactivation and a defect in the adaptation of RNase E concentration. These observations, coupled to the fact that RNase E overproduction slightly reduces the accumulation of the rpsO mRNA, suggest that this stabilization is caused in part by a limitation in RNase E concentration. An increase in the steady-state level of rpsT mRNA is also observed following a shift to 44 degrees C in minimal medium; however, processing of the 9 S rRNA precursor is not affected under these conditions. We thus propose that RNase E concentration changes in the cell in response to environmental conditions and that these changes can selectively affect the processing and the stability of individual mRNAs. Our data also indicate that the efficiency of cleavage of the rpsO mRNA by RNase E is modified by other factor(s) which remain to be identified.     

An easy cell-free protein synthesis system dependent on the addition of crude Escherichia coli tRNA

The protein-synthesizing S30 extract of Escherichia coli contains tRNA, which limits its applications in cell-free protein synthesis. Here, we show that at least Arg- and Ser-acceptor activities can be removed from a standard S30 extract by treatment with an immobilized RNase A resin. This RNase-treated extract exhibits no protein synthesis activity, but regains it when supplied with crude E. coli tRNA and a small amount of human placental RNase inhibitor. The protein synthesis is dependent on the addition of tRNA in the presence of the RNase inhibitor. Chloramphenicol acetyltransferase was synthesized with this system and found to be active.     

Rational immunotherapy with ribonuclease chimeras. An approach toward humanizing immunotoxins.

Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10(-7) M, whereas > 10(-4) M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly, the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates.     

Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

RNase L mediates transient control of the interferon response through modulation of the double-stranded RNA-dependent protein kinase PKR

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.