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Stability of the Sarcin/Ricin domain of 28S RNA in rat ribosome under hydrostatic pressure
Lü Biao,Li Qing,Liu Wangyi,Ruan Kangcheng
Chinese Science Bulletin , 1998, DOI: 10.1007/BF02884542
Abstract: Under 2 100 bar of hydrostatic pressure, KNA N-glycosidase can still cleave the N-C bond of adenosine at position 4324 in R/S domain of 28S RNA in rat ribosome, demonstrating that high pressure has no effect on the tertiary structure of S/R domain.
Stability of the Sarcin/Ricin domain of 28S RNA in rat ribosome under hydrostatic pressure

Lü Biao,Li Qing,Liu Wangyi,Ruan Kangcheng,

科学通报(英文版) , 1998,
Abstract: Under 2 100 bar of hydrostatic pressure, KNA N-glycosidase can still cleave the N-C bond of adenosine at position 4324 in R/S domain of 28S RNA in rat ribosome, demonstrating that high pressure has no effect on the tertiary structure of S/R domain.
大鼠核糖体28SRNA中Sarcin/Ricin结构域在高压力下的稳定性  [PDF]
吕飙,李清,刘望夷,阮康成
科学通报 , 1998,
Abstract: 在2100bar高压力下,大鼠核糖体28SRNA中的Sarcin/Ricin结构域是稳定的,同时还说明在高压力下,RNAN_糖苷酶仍保持水解Sarcin/Ricin结构域的活力.
Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity
Thibaut Pelat, Michael Hust, Martha Hale, Marie-Paule Lefranc, Stefan Dübel, Philippe Thullier
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-60
Abstract: In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity).Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication.Ricin, a 60 to 65 kDa glycoprotein derived from beans of the castor plant (Ricinus communis), is a lectin and member of the A-B toxin family. The B-chain carries the lectin function and binds to specific sugar residues of the target cell surface, allowing ricin to be internalized by endocytosis [1]. The A-chain (RCA-A) has RNA N-glycosidase activity, removing a highly conserved adenine residue in the sarcin/ricin loop of 28S rRNA. The RNA depurination in the ribosome inhibits docking of elongation factor 2, and prevents attachment of amino acids to the polypeptide chain. The result is irreversible inhibition of protein synthesis and eventual cell death [2]. Ricin is on the second priority list of the CDC and is regarded as a high risk for being utilised as a bioweapon.Ricin is hydrosoluble and, with an estimated LD5
alpha-Sarcin catalytic activity is not required for cytotoxicity
Spencer C Alford, Joel D Pearson, Amanda Carette, Robert J Ingham, Perry L Howard
BMC Biochemistry , 2009, DOI: 10.1186/1471-2091-10-9
Abstract: In this report, we assay α-sarcin cytotoxicity and ability to inhibit protein synthesis by direct cytoplasmic expression. We show that mutations in α-sarcin, which impair α-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate JNK, confirming that the sarcin-ricin loop remains intact and that the α-sarcin mutants are catalytically inactive. In addition, both mutant and wildtype variants of α-sarcin localize to the nucleus and cytoplasm, where they co-localize with ribosomal marker RPS6.We conclude that although protein synthesis inhibition likely contributes to cell death, it is not required. Thus, our results suggest that α-sarcin can promote cell death through a previously unappreciated mechanism that is independent of rRNA cleavage and JNK activation.α-Sarcin is a small fungal ribonuclease secreted by Aspergillus giganteus. It functions as a ribonuclease by catalytically cleaving a single phosphodiester bond in a well-defined RNA motif (the sarcin-ricin loop) within the rRNA scaffold of the large ribosomal subunit. This makes the ribosome unrecognizable to elongation factors and in turn, blocks protein synthesis [1]. In addition to its ability to inactivate the ribosome, α-sarcin selectively interacts with acidic phospholipids, and causes membrane fusion, as well as aggregation of artificial liposomes [2-4]. α-Sarcin's lipid binding properties allow for its uptake into the cell via acidic endosomes [4]. The mechanism of endosomal escape has not been characterized, but Golgi trafficking may be involved [5,6].α-Sarcin induces cell death through apoptosis but the mechanism is not completely understood [5]. It has been reported that the toxicity of α-sarcin is due to catalytic inactivation of the ribosome, and subsequent inhibition of protein synthesis [5]. Studies in which purified recombinant α-sarcin is added to the exterior of cells have supported this conclusion and suggested that cleavage of the sarcin
Inhibitors of the Cellular Trafficking of Ricin  [PDF]
Julien Barbier,Céline Bouclier,Ludger Johannes,Daniel Gillet
Toxins , 2012, DOI: 10.3390/toxins4010015
Abstract: Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress.
BiP Negatively Affects Ricin Transport  [PDF]
Tone F. Gregers,Sigrid S. Sk?nland,Sébastien W?lchli,Oddmund Bakke,Kirsten Sandvig
Toxins , 2013, DOI: 10.3390/toxins5050969
Abstract: The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.
The Need for Continued Development of Ricin Countermeasures  [PDF]
Ronald B. Reisler,Leonard A. Smith
Advances in Preventive Medicine , 2012, DOI: 10.1155/2012/149737
Abstract: Ricin toxin, an extremely potent and heat-stable toxin produced from the bean of the ubiquitous Ricinus communis (castor bean plant), has been categorized by the US Centers for Disease Control and Prevention (CDC) as a category B biothreat agent that is moderately easy to disseminate. Ricin has the potential to be used as an agent of biological warfare and bioterrorism. Therefore, there is a critical need for continued development of ricin countermeasures. A safe and effective prophylactic vaccine against ricin that was FDA approved for “at risk” individuals would be an important first step in assuring the availability of medical countermeasures against ricin. 1. Introduction In the aftermath of September 11, 2001, it has become increasingly clear that there is a need to enhance readiness against attack from both state sponsors and nonstate sponsors of bioterrorism. Ricin toxin, an extremely potent and heat-stable toxin produced from the bean of the Ricinus communis (castor bean plant) [1], has been categorized by the US Centers for Disease Control and Prevention (CDC) as a category B biothreat agent for biological warfare and bioterrorism [2]. In fact, according to Cookson and Nottingham, ricin was code named compound W and considered for weaponization during the US offensive Biological Warfare Program [3]. The US intelligence community believes that ricin was a component of the biowarfare program of the former Soviet Union, Iraq, and possibly other countries as well [4, 5]. Ricin toxin is relatively easy to produce and potentially lethal when delivered orally, intramuscularly, or through inhalation [4]. While the primary large-scale threat to US military personnel would be through powdered material that could be inhaled, ricin has been used successfully to assassinate individuals, to carry out suicide, and in 2003-2004, to terrorize US postal and Senate workers [4]. This paper reviews the rationale for development of ricin countermeasures and the progress toward achieving effective ricin countermeasures. 2. Background Ricin is a 65 kilodalton (kDa) polypeptide toxin comprised of two dissimilar polypeptide chains (an A-chain and a B-chain) held together by a disulfide bond [1, 4, 5]. The A-chain, ~32?kDa, targets the ribosome and is therefore a potent inhibitor of protein synthesis [4, 5]. Consequently, the A-chain has been classified as a ribosome-inactivating protein (RIP) [4, 5]. The B-chain, ~34?kDa, is a galactose or an N-acetylgalactosamine-binding lectin that attaches to cell-surface receptors [4, 5]. After binding and subsequent endocytosis,
Ricin Trafficking in Plant and Mammalian Cells  [PDF]
J. Michael Lord,Robert A. Spooner
Toxins , 2011, DOI: 10.3390/toxins3070787
Abstract: Ricin is a heterodimeric plant protein that is potently toxic to mammalian and many other eukaryotic cells. It is synthesized and stored in the endosperm cells of maturing Ricinus communis seeds (castor beans). The ricin family has two major members, both, lectins, collectively known as Ricinus communis agglutinin ll (ricin) and Ricinus communis agglutinin l (RCA). These proteins are stored in vacuoles within the endosperm cells of mature Ricinus seeds and they are rapidly broken down by hydrolysis during the early stages of post-germinative growth. Both ricin and RCA traffic within the plant cell from their site of synthesis to the storage vacuoles, and when they intoxicate mammalian cells they traffic from outside the cell to their site of action. In this review we will consider both of these trafficking routes.
Adapting Yeast as Model to Study Ricin Toxin A Uptake and?Trafficking  [PDF]
Bj?rn Becker,Manfred J. Schmitt
Toxins , 2011, DOI: 10.3390/toxins3070834
Abstract: The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S.?cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (?end3) and/or in RTA depurination activity on 28S rRNA (?rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.
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