A fluorescence anisotropy-based competition assay to identify inhibitors against ricin and Shiga toxin ribosome interactions.

Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

A Lipid Transfer Protein has Antifungal and Antioxidant Activity and Suppresses Fusarium Head Blight Disease and DON Accumulation in Transgenic Wheat.

Trichothecene mycotoxins such as deoxynivalenol (DON) are virulence factors of Fusarium graminearum, which causes Fusarium head blight, one of the most important diseases of small grain cereals. We previously identified a nonspecific lipid transfer protein (nsLTP) gene, AtLTP4.4, which was overexpressed in an activation-tagged Arabidopsis line resistant to trichothecin, a type B trichothecene in the same class as DON. Here we show that overexpression of AtLTP4.4 in transgenic wheat significantly reduced F. graminearum growth in 'Bobwhite' and 'RB07' lines in the greenhouse and reduced fungal lesion size in detached leaf assays. Hydrogen peroxide accumulation was attenuated on exposure of transgenic wheat plants to DON, indicating that AtLTP4.4 may confer resistance by inhibiting oxidative stress. Field testing indicated that disease severity was significantly reduced in two transgenic 'Bobwhite' lines expressing AtLTP4.4. DON accumulation was significantly reduced in four different transgenic 'Bobwhite' lines expressing AtLTP4.4 or a wheat nsLTP, TaLTP3, which was previously shown to have antioxidant activity. Recombinant AtLTP4.4 purified from Pichia pastoris exhibited potent antifungal activity against F. graminearum. These results demonstrate that overexpression of AtLTP4.4 in transgenic wheat suppresses DON accumulation in the field. Suppression of DON-induced reactive oxygen species by AtLTP4.4 might be the mechanism by which fungal spread and mycotoxin accumulation are inhibited in transgenic wheat plants.

Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress-induced HAC1 mRNA splicing on the ribosome.

Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with ribosomes.

Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes.

Trichothecene mycotoxins are natural contaminants of small grain cereals and are encountered in the environment, posing a worldwide threat to human and animal health. Their mechanism of toxicity is poorly understood, and little is known about cellular protection mechanisms against trichothecenes. We previously identified inhibition of mitochondrial protein synthesis as a novel mechanism for trichothecene-induced cell death. To identify cellular functions involved in trichothecene resistance, we screened the Saccharomyces cerevisiae deletion library for increased sensitivity to nonlethal concentrations of trichothecin (Tcin) and identified 121 strains exhibiting higher sensitivity than the parental strain. The largest group of sensitive strains had significantly higher reactive oxygen species (ROS) levels relative to the parental strain. A dose-dependent increase in ROS levels was observed in the parental strain treated with different trichothecenes, but not in a petite version of the parental strain or in the presence of a mitochondrial membrane uncoupler, indicating that mitochondria are the main site of ROS production due to toxin exposure. Cytotoxicity of trichothecenes was alleviated after treatment of the parental strain and highly sensitive mutants with antioxidants, suggesting that oxidative stress contributes to trichothecene sensitivity. Cotreatment with rapamycin and trichothecenes reduced ROS levels and cytotoxicity in the parental strain relative to the trichothecene treatment alone, but not in mitophagy deficient mutants, suggesting that elimination of trichothecene-damaged mitochondria by mitophagy improves cell survival. These results reveal that increased mitophagy is a cellular protection mechanism against trichothecene-induced mitochondrial oxidative stress and a potential target for trichothecene resistance.

A genome-wide screen in Saccharomyces cerevisiae reveals a critical role for the mitochondria in the toxicity of a trichothecene mycotoxin.

Trichothecene mycotoxins synthesized by Fusarium species are potent inhibitors of eukaryotic translation. They are encountered in both the environment and in food, posing a threat to human and animal health. They have diverse roles in the cell that are not limited to the inhibition of protein synthesis. To understand the trichothecene mechanism of action, we screened the yeast knockout library to identify genes whose deletion confers resistance to trichothecin (Tcin). The largest group of resistant strains affected mitochondrial function, suggesting a role for fully active mitochondria in trichothecene toxicity. Tcin inhibited mitochondrial translation in the wild-type strain to a greater extent than in the most resistant strains, implicating mitochondrial translation as a previously unrecognized site of action. The Tcin-resistant strains were cross-resistant to anisomycin and chloramphenicol, suggesting that Tcin targets the peptidyltransferase center of mitochondrial ribosomes. Tcin-induced cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular morphology of mitochondria. Treatment of yeast cells with Tcin led to the fragmentation of the tubular mitochondrial network, supporting a role for Tcin in disruption of mitochondrial membrane morphology. These results provide genome-wide insight into the mode of action of trichothecene mycotoxins and uncover a critical role for mitochondrial translation and membrane maintenance in their toxicity.

Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin.

Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

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.

Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach.

Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

Functional reversion to identify controlling genes in multigenic responses: analysis of floral abortion.

In many situations, organisms respond to stimuli by altering the activity of large numbers of genes. Among these, certain ones are likely to control the phenotype while others play a secondary role or are passively altered without directly affecting the phenotype. Identifying the controlling genes has proven difficult. However, in a few instances, it has been possible to reverse the phenotype by physiological or biochemical means without altering the genetics of the organism. During this functional reversion, only a few genes may respond, thus identifying those likely to be controlling the phenotype. Floral abortion during a water shortage in maize is an example because the response is inherently multigenic, and the phenotype can be reversed by physiological/biochemical means. A recent analysis used this reversal to reveal that only a few genes are likely to control the abortion phenotype. In maize, these genes coded for a cell wall invertase (Incw2), a soluble invertase (Ivr2), a ribosome-inactivating protein (RIP2), and phospholipase D (PLD1). The invertases appeared to control the normal sugar uptake by the ovaries. Their down-regulation depleted ovary sugar pools and resulted in an up-regulation of the genes for ribosome-inactivating protein and for phospholipase. The latter changes appeared to initiate senescence that degraded cell membranes, thus causing irreversible abortion. With these findings, these genes have become targets for preventing abortion. This approach might have value in other contexts with some additional methods.

Imaging and quantifying carbohydrate transport to the developing ovaries of maize.

BACKGROUND AND AIMS: Shade or inadequate water can inhibit photosynthesis and limit the development of maize (Zea mays) ovaries around the time of pollination, potentially reducing the number of kernels at harvest. This study investigated whether the decreased photosynthesis diminished only the sugar supply or also altered the transport path to the ovaries. METHODS: Photosynthesis and water potentials (Psiw) were measured in the leaves while dry matter delivery was monitored in the ovaries. Ovary glucose, starch and acid invertase activities were measured in situ. Stems were fed xylem-mobile safranin or phloem-mobile carboxyfluorescein (CF), and the dye transport to the ovaries was determined. KEY RESULTS: Under normal conditions, the ovaries gained in dry mass, and starch accumulated in the pedicel and ovary wall. Glucose accumulated in the pedicel, apparently in the apoplast where insoluble (cell-wall-bound) acid invertase acted on the arriving sucrose. A glucose gradient developed from pedicel to nucellus. Safranin moved in the xylem and did not reach the ovary, but CF moved in the phloem and arrived at the ovary. CF also spread into the pedicel but unlike glucose it did not enter the nucellus. Low Psiw or shade decreased leaf photosynthesis, ovary dry mass accumulation, invertase activities, pedicel glucose, starch accumulation and CF delivery. Removal of these treatments reversed the effects. CONCLUSIONS: The success of CF in tracing the general path and rate of carbohydrate transport gave visual evidence that phloem transport to the ovary decreased at low Psiw or in the shade but otherwise remained functional. The decreases indicated that losses in carbohydrate delivery are central features of failed ovary development under these conditions. The selectivity of transport into the nucellus resembled the situation later when embryo and endosperm are present and selective uptake occurs from the apoplast.

Glucose localization in maize ovaries when kernel number decreases at low water potential and sucrose is fed to the stems.

BACKGROUND AND AIMS: Around the time of anthesis, young ovary development in maize (Zea mays) is vulnerable to 2 or 3 d of water deficits that inhibit photosynthesis. Abortion can result, and fewer kernels are produced. A breakdown of stored ovary starch is associated with the abortion and was investigated in the present study by localizing the breakdown product glucose in the ovaries. METHODS: Ovary glucose was localized with fluorescent Resorufin. Insoluble invertase was localized in vivo and soluble invertase in situ. Sucrose was infused into the stems to vary the sugar flux to the ovaries. KEY RESULTS: At high water potential (high Psi(w)), photosynthesis was rapid in the parent. The upper pedicel of the ovaries had a high activity of insoluble acid invertase and a large amount of glucose and starch. Because the invertase was wall-bound, sucrose hydrolysis appeared to occur in the pedicel apoplast. Soluble invertase was undetected inside the pedicel cells but was present in the nucellus cells where low concentrations of glucose occurred. This created a glucose gradient between pedicel and nucellus that favoured glucose uptake by the developing ovary. At low Psi(w), photosynthesis was inhibited, pedicel glucose and starch were depleted, the glucose gradient became negligible, and abortion occurred. When sucrose was fed, glucose, starch and the glucose gradient were maintained somewhat and were normally distributed in the ovaries. Abortion was diminished. CONCLUSIONS: The apoplast hydrolysis of sucrose unloaded from phloem is similar to that described by others during later development when embryo and endosperm are present and separated from the parent by an apoplast. The disappearance of the glucose gradient at low Psi(w) may have inhibited glucose movement into the ovary. The low glucose in the ovaries may have a role in the abortion response.

Sugar-responsive gene expression, invertase activity, and senescence in aborting maize ovaries at low water potentials.

BACKGROUND AND AIMS: Ovary abortion can occur in maize (Zea mays) if water deficits lower the water potential (psiw) sufficiently to inhibit photosynthesis around the time of pollination. The abortion decreases kernel number. The present work explored the activity of ovary acid invertases and their genes, together with other genes for sucrose-processing enzymes, when this kind of abortion occurred. Cytological evidence suggested that senescence may have been initiated after 2 or 3 d of low psiw, and the expression of some likely senescence genes was also determined. METHODS: Ovary abortion was assessed at kernel maturity. Acid invertase activities were localized in vivo and in situ. Time courses for mRNA abundance were measured with real time PCR. Sucrose was fed to the stems to vary the sugar flux. KEY RESULTS: Many kernels developed in controls but most aborted when psiw became low. Ovary invertase was active in controls but severely inhibited at low psiw for cell wall-bound forms in vivo and soluble forms in situ. All ovary genes for sucrose processing enzymes were rapidly down-regulated at low psiw except for a gene for invertase inhibitor peptide that appeared to be constitutively expressed. Some ovary genes for senescence were subsequently up-regulated (RIP2 and PLD1). In some genes, these regulatory changes were reversed by feeding sucrose to the stems. Abortion was partially prevented by feeding sucrose. CONCLUSIONS: A general response to low psiw in maize ovaries was an early down-regulation of genes for sucrose processing enzymes followed by up-regulation of some genes involved in senescence. Because some of these genes were sucrose responsive, the partial prevention of abortion with sucrose feeding may have been caused in part by the differential sugar-responsiveness of these genes. The late up-regulation of senescence genes may have caused the irreversibility of abortion.