Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin

Ricin A-chain, abrin A-chain, and alpha-trichosanthin are members of a larger group of proteins called ribosome-inactivating proteins. These proteins all function to catalytically inactivate eukaryotic 60 S ribosomal subunits leading to rapid shutdown of protein synthesis. They are homologous in sequence and are probably evolutionarily related. We have determined the complete primary amino acid sequence of alpha-trichosanthin and have found it to be homologous, as expected, to that of abrin A-chain and ricin A-chain. A crystal structure for ricin, which includes ricin A-chain and ricin B-chain, has been determined from x-ray diffraction data. Based on the sequence homologies of these proteins, we fit the primary sequences of abrin A-chain and alpha-trichosanthin to the backbone structure for ricin A-chain and have generated energy-minimized molecular models for them. These models should prove useful in studying the structural-functional relationships of these proteins in particular and of the class in general.     

Differential Effect of Ribosome-Inactivating Proteins on Plant Ribosome Activity and Plant Cells Growth

The effect of ribosome-inactivating proteins (RIPs), eithersingle-chain or toxins, was studied on plant ribosomes. RIPsdid not affect ribosomes from their own plants, while inhibitingto a variable extent protein synthesis by heterologous plantribosomes. Ricin stimulated and PAP—S inhibited the growthof carrot cells in culture. Key words: Plant ribosomes, Ribosome-inactivating proteins, Protein synthesis, Ribosome specificity, Plant cell cultures     

Microinjection of ricin entrapped in unilamellar liposomes into a ricin-resistant mutant of baby hamster kidney cells

Fluorescein-labelled Ricin was entrapped in unilamellar liposomes; 14 μg of protein was entrapped by 1 mg of lipids. Liposomes added to cells in culture in low serum medium can deliver entrapped Ricin to a Ricin-resistant mutant of baby hamster kidney(BHK)cells. Ricin entrapped in unilamellar liposomes inhibits protein biosynthesis at a concentration of 1.75 μg/ml in Ricin-resistant cells. Ricin dissolved in medium at 50 μg/ml does not affect protein synthesis in these cells.     

Microinjection of ricin entrapped in unilamellar liposomes into a ricin-resistant mutant of baby hamster kidney cells.

Fluorescein-labelled Ricin was entrapped in unilamellar liposomes; 14 microgram of protein was entrapped by 1 mg of lipids. Liposomes added to cells in culture in low serum medium can deliver entrapped Ricin to a Ricin-resistant mutant of baby hamster kidney(BHK)cells. Ricin entrapped in unilamellar liposomes inhibits protein biosynthesis at a concentration of 1.75 microgram/ml in Ricin-resistant cells. Ricin dissolved in medium at 50 microgram/ml does not affect protein synthesis in these cells.     

Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli (Short Communication)

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.     

The role of calcium ions for the expression of ricin toxicity in cultured macrophages

Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited ³H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca²⁺-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg²⁺ did not affect protein synthesis or binding of ¹²⁵I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages.     

核糖体RNA拓扑学与RNAN－糖苷酶研究进展（上）

核糖体ＲＮＡ拓扑学的研究对阐明核糖体ＲＮＡ（ｒＲＮＡ）在蛋白质生物合成中的作用具有重要的意义。ＲＮＡＮ－糖苷０酶是一类核糖体失活蛋白．它只水解ｒＲＮＡ特定位置上一个腺苷酸的糖苷键,释放一个腺嘌呤碱基,使核糖体失活。ＲｉｃｉｎＡ链是研究得最早和最详细的ＲＮＡＮ－糖苷酶,迄今已发现有二十五种核糖体失活蛋白具有ＲＮＡＮ－糖苷酶活性。ＲＮＡＮ－糖苷酶作用于２８ＳｒＲＮＡ的α－ｓａｒｃｉｎ结构域,改变核糖体的构象而使其失活。     

Effect of ricin on acute phase response in rats.

Ricin, a highly toxic protein from castor beans was administered (ip) to rats in a dose of 1.25 micrograms/100 g to selectively deplete at least 60-70% of Kupffer cells. This dose spared hepatocytes. This rat model was used to study acute phase protein synthesis and the role of Kupffer cells in acute phase response (APR). Ricin itself induced an APR, similar in pattern but of lower magnitude, than that induced by turpentine. However, the effect of combination of ricin and turpentine on APR was not additive. Kupffer cells appear to play permissive role in APR through mediators like hepatocytes stimulating factors.     

核糖体RNA拓扑学与RNA N－糖苷酶研究进展（上）

核糖体RNA拓扑学的研究对阐明核糖体RNA(rRNA)在蛋白质生物合成中的作用具有重要的意义.RNA N-糖苷酶是一类核糖体失活蛋白.它只水解rRNA特定位置上一个腺苷酸的糖苷键,释放一个腺嘌呤碱基,使核糖体失活.Ricin A链是研究得最早和最详细的RNA N-糖苷酶,迄今已发现有二十五种核糖体失活蛋白具有RNA N-糖苷酶活性.RNA N-糖苷酶作用于28S rRNA的α-sarcin结构域,改变核糖体的构象而使其失活.     

Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli

Ricin and its A chains can be used to conjugate with monoclonal antibodies to prepare immunotoxins. Ricin A chain (RTA) and its modification RTA-KDEL (ER-retrieval signal) were expressed with the pKK223.3 system in Escherichia coli under control of a tac promoter. The recombinant proteins can be purified by one-step affinity chromatography on a column of Blue-Sepharose 6B. The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF, and ECV-304 cells following fluid-phase endocytosis. RTA-KDEL was somewhat more cytotoxic than RTA itself in the different cell lines. The results suggest that rRTA-KDEL may be useful for the synthesis of more potent immunotoxins.     

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.     

Immunoglobulin synthesis by free polysomes of mouse myeloma cells

Free polyribosomes isolated from mouse myeloma cells in tissue culture synthesize immunoglobulin chains. The presence of these peptide chains in the cytoplasm of intact myeloma cells has been investigated. Some immunoglobulin chains were observed, but it could not be ruled out that these were originally inside cisternae of the endoplasmic reticulum, which were broken during hogenization. We have also investigated the transport of the hypothetical cytoplastic immunoglobulins into the cisternae of the endoplasmic reticulum after incubation with radioactive amino acids and subsequent chase in the absence of protein synthesis. A model to account for synthesis of immunoglobulins on free polysomes is presented. This model assigns specificity for translation on membrane-bound polysomes to the N-terminal region of secretory proteins.     

Cluster analysis of aminoacyl-tRNAs from mouse plasmacytomas correlates chromatographic profiles with myeloma protein similarity,clonal origin of tumor lines,and the neoplastic nature of the tissues

In order to test whether tRN A populations are correlated with (determined by or adapted to) the major proteins synthesized by tumor cells, RPC-5 Chromatographie profiles of aminoacyl-tRNAs from 11 mouse plasmacytomas and from normal adult mouse liver and brain were analyzed by the use of “dissimilarity indices” drawn from all possible pairs of tissues. Cluster analysis was then performed and dendrograms constructed. Although myeloma protein synthesis is only one of many proteins being synthesized by these malignant cells, a novel nonparametric statistical analysis of these dendrograms indicates that independently arising tumors have more similar profiles if their immunoglobulin light and heavy chains are very similar than if these chains are dissimilar (P < 0·015). Even more strikingly significant was the finding that drastic changes in myeloma protein synthesis such as loss of both heavy and light chain synthesis do not result in increased dissimilarity of aminoacyl-tRNA profiles (P < 0·00001). Unlike other eukaryotic systems such as sheep reticulocytes and silk worm silk gland which have been shown to adapt their tRNA populations to changes in protein synthesis, these plasmacytomas do not appear to do so.The novel use of statistical methods, esp. cluster analysis, to examine graphic displays of data may have useful applications in comparing other Chromatographie profiles, densitometric scans, etc.     

Effects of ricin on the ribosomal sites involved in the interaction of the elongation factors.

The effects of ricin on the different steps of the elongation cycle of protein synthesis in a rabbit reticulocyte cell-free system are studied in this paper. The toxin most probably acts by catalytically inactivating the ribosomes, since a single molecule of the toxin can inactivate 300 ribosomes for poly(U)-directed phenylalanine incorporation. The effect of the toxin on the ribosome is irreversible. Ricin specifically inhibits elongation-factor-1-dependent aminoacyl-tRNA binding to ribosomes but has no effect on the non-enzymic binding of aminoacyl-tRNA. Ricin also inhibits formation of the complex elongation-factor-2 - ribosome - nucleotide with GTP, GDP or GMP-P(CH2)P. However, the toxin has no effect on translocation. These apparently conflicting results are discussed in this study.     

RICIN-BINDING PROTEINS ALONG THE ENDOCYTIC PATHWAY: THE MAJOR ENDOSOMAL RICIN-BINDING PROTEIN IS ENDOSOME-SPECIFIC

Ricin is internalized after binding at the cell surface via lectin activity of the B-chain recognizing terminal galactose residues. Ricin-A chain is then translocated to the cytosol from various endocytic structures. Cell death is the result of catalytic inactivation of protein synthesis. Using ¹²⁵I-ricin overlays, we examined the distribution of ricin binding-proteins within highly purified preparations of plasma membrane vesicles, endosomes and lysosomes from lymphocytes. All compartments of the endocytic pathway had distinct profiles; some ricin-binding proteins were present throughout the pathway; others were restricted to the plasma membrane and endosomes. The major endosomal protein recognized by ¹²⁵I-ricin, a 166kDa glycoprotein, was endosome-specific. When endosomal proteins were solubilized before chromatography onto ricin-agarose this protein was also by far the major specifically-bound glycoprotein. This 166 kDa glycoprotein might be involved in ricin translocation from this compartment.     

Inhibition of Fatty Acid Metabolism Reduces Human Myeloma Cells Proliferation

Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40–70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.     

Further studies on eukaryote DNA stimulation of amino acid incorporation in E. coli extracts

DNA from adenovirus-2 and mouse myeloma tumors stimulate RNA synthesis and amino acid incorporation into protein in a cell-free extract from Escherichia coli. The RNA synthesis is dependent on exogenous DNA, and the RNA can be hybridized to respective template DNA. A major part of this RNA is also found attached to E. coli polysomes suggesting that RNA with messenger-like activity has been synthesized. However, the in vitro-synthesized polypeptides using adenovirus DNA or myeloma DNA do not correspond in size or antigenic activity to either the virion proteins or immunoglobulins, respectively.     

The enemy within: ricin and plant cells

Ricin, a ribosome-inactivating protein from the seeds of the castor oil plant (Ricinus communis L.) is one of the most potent cell poisons known. It is able to bind and enter most mammalian cells where it exploits their fully reversible secretory pathway to reach the endoplasmic reticulum. Ricin is then able to exit the endoplasmic reticulum to access the cytosol where it inhibits protein synthesis, thus killing the cells. Castor bean ribosomes are sensitive to ricin, but the plant has developed strategies to protect its own cells from suicide. The intracellular routing of ricin has been traditionally studied by exogenously adding toxin to mammalian cells and by following its path through the cell. However, the extreme potency of this protein has prevented the final membrane transport step from being studied in detail. Now, the expression of ricin in heterologous plant cells is providing helpful in elucidating details of both toxin biosynthesis and vacuolar targeting, and in studying membrane translocation of the catalytic subunit from the endoplasmic reticulum to the cytosol.Keywords: Ricin, ribosome-inactivating protein, castor oil plant, seeds, inhibitor, membrane transport.      

Identification of the ricin lipase site and implication in cytotoxicity

Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its N-glycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.     

Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

Background  

Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.