Trichothecene Mycotoxins Inhibit Mitochondrial Translation—Implication for the Mechanism of Toxicity

Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes

Do the A Subunits Contribute to the Differences in the Toxicity of Shiga Toxin 1 and Shiga Toxin 2?

Shiga toxin producing Escherichia coli O157:H7 (STEC) is one of the leading causes of food-poisoning around the world. Some STEC strains produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or variants of either toxin, which are critical for the development of hemorrhagic colitis (HC) or hemolytic uremic syndrome (HUS). Currently, there are no therapeutic treatments for HC or HUS. E. coli O157:H7 strains carrying Stx2 are more virulent and are more frequently associated with HUS, which is the most common cause of renal failure in children in the US. The basis for the increased potency of Stx2 is not fully understood. Shiga toxins belong to the AB5 family of protein toxins with an A subunit, which depurinates a universally conserved adenine residue in the α-sarcin/ricin loop (SRL) of the 28S rRNA and five copies of the B subunit responsible for binding to cellular receptors. Recent studies showed differences in the structure, receptor binding, dependence on ribosomal proteins and pathogenicity of Stx1 and Stx2 and supported a role for the B subunit in differential toxicity. However, the current data do not rule out a potential role for the A1 subunits in the differential toxicity of Stx1 and Stx2. This review highlights the recent progress in understanding the differences in the A1 subunits of Stx1 and Stx2 and their role in defining toxicity

Arabidopsis Bax Inhibitor-1 inhibits cell death induced by pokeweed antiviral protein in Saccharomyces cerevisiae

WOS: 000218676600003PubMed ID: 28357275Apoptosis is an active form of programmed cell death (PCD) that plays critical roles in the development, differentiation and resistance to pathogens in multicellular organisms. Ribosome inactivating proteins (RIPs) are able to induce apoptotic cell death in mammalian cells. In this study, using yeast as a model system, we showed that yeast cells expressing pokeweed antiviral protein (PAP), a single-chain ribosome-inactivating protein, exhibit apoptotic-like features, such as nuclear fragmentation and ROS production. We studied the interaction between PAP and AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1), a plant anti-apoptotic protein, which inhibits Bax induced cell death. Cells expressing PAP and AtBI-1 were able to survive on galactose media compared to PAP alone, indicating a reduction in the cytotoxicity of PAP in yeast. However, PAP was able to depurinate the ribosomes and to inhibit total translation in the presence of AtBI-1. A C-terminally deleted AtBI-1 was able to reduce the cytotoxicity of PAP. Since anti-apoptotic proteins form heterodimers to inhibit the biological activity of their partners, we used a coimmunoprecipitation assay to examine the binding of AtBI-1 to PAP. Both full length and C-terminal deleted AtBI-1 were capable of binding to PAP. These findings indicate that PAP induces cell death in yeast and AtBI-1 inhibits cell death induced by PAP without affecting ribosome depurination and translation inhibition.National Science FoundationNational Science Foundation (NSF) [MCB-0348299, MCB-0130531]This work was supported by National Science Foundation grants MCB-0348299 and MCB-0130531 to Nilgun E. Tumer. We are grateful to Dr. Ozlem Tuncay for her help in statistical analyses

An N-Terminal Fragment of Yeast Ribosomal Protein L3 Inhibits the Cytotoxicity of Pokeweed Antiviral Protein in Saccharomyces cerevisiae

We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of yeast L3 (L3Δ99) with PAP in transgenic tobacco plants completely abolished the toxicity of PAP. In this study, we investigated the interaction between PAP and L3Δ99 in Saccharomyces cerevisiae. Yeast cells co-transformed with PAP and L3Δ99 showed markedly reduced growth inhibition and reduced rRNA depurination by PAP, compared to cells transformed with PAP alone. Co-transformation of yeast with PAP and L3Δ21 corresponding to the highly conserved N-terminal 21 amino acids of L3Δ99, reduced the cytotoxicity of PAP. PAP mRNA and protein levels were elevated and L3Δ99 or L3Δ21 mRNA and protein levels were reduced in yeast co-transformed with PAP and L3Δ99 or with PAP and L3Δ21, respectively. PAP interacted with L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that the interaction between L3Δ21 and PAP may inhibit PAP-mediated depurination of the SRL, leading to a reduction in the cytotoxicity of PAP

Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding

Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTA–ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTA–ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved α-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin