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Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. [ABSTRACT FROM AUTHOR]

Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome. [ABSTRACT FROM AUTHOR]

Ricin A-chain ( RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α- P2β and P2α- P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the sarcin-ricin loop. Using small interfering RNA-mediated knockdown of P1/ P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/ P2-depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1- P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes. Structured digital abstract P2, P1 and RTA physically interact by surface plasmon resonance (View interaction), P2, P1 and RTA physically interact by anti bait coimmunoprecipitation (View interaction) [ABSTRACT FROM AUTHOR]

Abstract: Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2βin vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1. [Copyright &y& Elsevier]

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity. [ABSTRACT FROM AUTHOR]

Ribosome-inactivating proteins (RIPs) are potent toxins that inactivate ribosomes by catalytically removing a specific adenine from the α-sarcin/ricin loop (SRL) of the large rRNA. Direct assays for measuring depurination activity and indirect assays for measuring the resulting translation inhibition have been employed to determine the enzyme activity of RIPs. Rapid and sensitive methods to measure the depurination activity of RIPs are critical for assessing their reaction mechanism, enzymatic properties, interaction with ribosomal proteins, ribotoxic stress signaling, in the search for inhibitors and in the detection and diagnosis of enteric infections. Here, we review the major assays developed for measuring the catalytic activity of RIPs, discuss their advantages and disadvantages and explain how they are used in understanding the catalytic mechanism, ribosome specificity, and dynamic enzymatic features of RIPs. [ABSTRACT FROM AUTHOR]

Abstract: The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination. [Copyright &y& Elsevier]

The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near-perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (V H Hs) previously reported to recognize epitopes in proximity to RTA's active site. Three of the V H Hs, V2A11, V8E6, and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies ("intrabodies"). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTA's active site and interact with key catalytic residues. V2G10, by contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four V H Hs also penetrated/occluded RTA's active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.toxin, antibody, structure, intracellular Image 1 • Ricin's catalytic subunit (RTA) inactivates ribosome with high efficiency. • Alpaca single-domain antibodies (VHH) that target RTA's active site identified. • Intracellular expression (intrabody) of VHHs neutralizes ricin toxin. • Seven crystal structures of VHH-RTA complexes reported. • Results pave the way for novel antitoxin therapies. [ABSTRACT FROM AUTHOR]