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RNA
Published in Paul Pumpens, Single-Stranded RNA Phages, 2020
The MS2 RNA contributed as a substrate to the evaluation of the pokeweed antiviral protein (PAP), that was isolated from the leaves of the pokeweed plant Phytolacca americana and characterized initially as a ribosome-inactivating protein (Rajamohan et al. 1999). The PAP acted as the site-specific RNA N-glycosidase and removed catalytically a single adenine base from a highly conserved loop of the large rRNA species in eukaryotic and prokaryotic ribosomes. In contrast to ricin A chains, the PAP-I, PAP-II, and PAP-III isoforms demonstrated ability to depurinate not only MS2, but also HIV-1 RNA, and appeared as potent anti-HIV agents (Rajamohan et al. 1999).
The Structural Biology, Biochemistry, Toxicology, and Military Use of the Ricin Toxin and the Associated Treatments and Medical Countermeasures for Ricin Exposure
Published in Brian J. Lukey, James A. Romano, Salem Harry, Chemical Warfare Agents, 2019
Terry J. Henderson, George Emmett, Russell M. Dorsey, Charles B. Millard, Ross D. LeClaire, Harry Salem
Catalytic RTA recognizes and binds a highly conserved region in the large 28S rRNA referred to as the sarcin/ricin loop (Rajamohan et al., 2001), a very short stem-loop structure in domain VII of the rRNA molecule ~400 nucleotides from its 3'-end. Within the stem-loop, the ring of a single adenine nucleotide, A4324, becomes intercalated between the aromatic rings of Y80 and Y123 in the catalytic cleft of RTA (see Figure 13.4a) and is hydrolyzed at the carbon–nitrogen glycosidic bond by N-glycosidase action (Endo et al., 1987). Although the detailed mechanism of this event is not known, Monzingo and Robertus (1992) have proposed a mechanism based on their high-resolution X-ray crystallographic data of substrate analogs in the RTA active site. As summarized in Figure 13.6, the mechanism begins with the sarcin–ricin loop substrate binding the RTA active site with the A4324 ring system intercalating between the aromatic rings of Y80 and Y123 (Figure 13.4a). R180 donates a proton to N-3 of the A4324 ring system to break the bond between N-9 of the ring system and C-1′ of the A4324 ribose (Figure 13.6a). This bond cleavage leaves cation character on the ribose ring in the form of an oxycarbonium ion and gives anion character to the A4324 ring system (Figure 13.6b); the oxycarbonium ion should have sp2 character at C-1′ (indicated in Figure 13.6b by the partial double-bond character with O-4′). The bond-forming process (represented in the path between Figure 13.6b and c) begins with R180 donating a proton to N-3 of the adenine ring system to increase the basicity of the side chain. This, in turn, acts to pull a proton from a water molecule, creating hydroxide character to assist solvent attack on ribose and ultimately, a neutral ribose (Figure 13.6c). The figure shows the A4324 product with hydrogen at N-3, but this will readily tautomerize in solution to the more stable form with hydrogen at N-9. Site-specific RNA N-glycosidase activity is a feature common to all previously identified type I and II RIP toxins (Endo et al., 1987). The activity prevents the binding of elongation factors EF-1 and EF-2, resulting in the cessation of mRNA translation.
Immunotoxins and nanobody-based immunotoxins: review and update
Published in Journal of Drug Targeting, 2021
Mohammad Reza Khirehgesh, Jafar Sharifi, Fatemeh Safari, Bahman Akbari
RT is a heterodimer glycoprotein in the seeds of the Ricinus communis that isolated first by Peter Hermann Stillmark [92]. RT (∼64 kDa) belongs to the type II ribosome-inactivating proteins (RIP II) family that consists of A chain, enzymatically active subunit (RTA), and B chains, cell binding subunit (RTB). RTA composes of 267 amino acids (∼32 kDa) with irreversible RNA N-glycosidase activity. RTA has been used in IT construction as a cytotoxic moiety [93–95]. The subunit prevents the binding of eEF2 to the ribosome by depurination of adenine 4324 at the specific alpha-sarcin/ricin loop in 28 s ribosomal RNA (rRNA). Subsequently, protein synthesis ceases at the translocation step and the cells die apoptotically [96,97]. RTB is a galactose-binding lectin (GBL) that comprises 262 amino acids. RTA and RTB connect with interchain disulphide bonds in Cys-259 of RTA and Cys-4 of RTB (∼34 kDa) [98,99]. The binding of RTB to β1-4-linked galactose on the eukaryotic cells surface leads to clathrin-mediated endocytosis or clathrin-independent endocytosis of the toxin [100,101]. After processing in the endosome and Golgi network, RT enters the ER lumen. In the ER lumen, PDI reduces the disulphide bond between RTA and RTB, leads to the separation of the two subunits. Finally, RTA transfers to the cytoplasm and inhibits protein synthesis by inactivating ribosomes, so leads to cell death [102] (Figure 4).