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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.
John E. McLaughlin; Noura I. Darwish; Jeffrey Garcia-Sanchez; Neerja Tyagi; Harold N. Trick; Susan McCormick; Ruth Dill-Macky; Nilgun E. Tumer. A Lipid Transfer Protein has Antifungal and Antioxidant Activity and Suppresses Fusarium Head Blight Disease and DON Accumulation in Transgenic Wheat. Phytopathology® 2021, 111, 671 -683.
AMA StyleJohn E. McLaughlin, Noura I. Darwish, Jeffrey Garcia-Sanchez, Neerja Tyagi, Harold N. Trick, Susan McCormick, Ruth Dill-Macky, Nilgun E. Tumer. A Lipid Transfer Protein has Antifungal and Antioxidant Activity and Suppresses Fusarium Head Blight Disease and DON Accumulation in Transgenic Wheat. Phytopathology®. 2021; 111 (4):671-683.
Chicago/Turabian StyleJohn E. McLaughlin; Noura I. Darwish; Jeffrey Garcia-Sanchez; Neerja Tyagi; Harold N. Trick; Susan McCormick; Ruth Dill-Macky; Nilgun E. Tumer. 2021. "A Lipid Transfer Protein has Antifungal and Antioxidant Activity and Suppresses Fusarium Head Blight Disease and DON Accumulation in Transgenic Wheat." Phytopathology® 111, no. 4: 671-683.
The principal virulence factor of human-pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome binding site is exposed. We show here that Stx2a binds to an 11mer peptide (P11) mimicking the CTD of P stalk proteins with low micromolar affinity. We cocrystallized Stx2a with P11 and defined their interactions by X-ray crystallography. We found that the last six residues of P11 inserted into a shallow pocket on Stx2A1 and interacted with Arg172, Arg176 and Arg179, which were previously shown to be critical for binding of Stx2A1 to the ribosome. Stx2a formed a distinct P11 binding mode within a different surface pocket relative to ricin toxin A subunit and trichosanthin, suggesting different ribosome recognition mechanisms for each ribosome inactivating protein (RIP). The binding mode of Stx2a to P11 is also conserved among the different Stx subtypes. Furthermore, the P stalk protein CTD is flexible and adopts distinct orientations and interaction modes depending on the structural differences between the RIPs. Structural characterization of the Stx2a-ribosome complex is important for understanding the role of the stalk in toxin recruitment to the sarcin/ricin loop and may provide a new target for inhibitor discovery.
Michael J. Rudolph; Simon A. Davis; Nilgun E. Tumer; Xiao-Ping Li. Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins. Journal of Biological Chemistry 2020, 295, 15588 -15596.
AMA StyleMichael J. Rudolph, Simon A. Davis, Nilgun E. Tumer, Xiao-Ping Li. Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins. Journal of Biological Chemistry. 2020; 295 (46):15588-15596.
Chicago/Turabian StyleMichael J. Rudolph; Simon A. Davis; Nilgun E. Tumer; Xiao-Ping Li. 2020. "Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins." Journal of Biological Chemistry 295, no. 46: 15588-15596.
Ricin toxin A subunit (RTA) removes an adenine from the universally conserved sarcin/ricin loop (SRL) on eukaryotic ribosomes thereby inhibiting protein synthesis. No high affinity and selective small-molecule therapeutic antidotes have been reported against ricin toxicity. RTA binds to the ribosomal P stalk to access the SRL. The interaction anchors RTA to the P protein C-termini at a well-defined hydrophobic pocket, which is on the opposite face relative to the active site. The RTA ribosome binding site has not been previously targeted by small molecule inhibitors. We used fragment screening with surface plasmon resonance to identify small-molecular-weight-lead compounds that bind RTA and defined their interactions by crystallography. We identified five fragments, which bound RTA with mid-micromolar affinity. Three chemically distinct binding fragments were co-crystallized with RTA and crystal structures were solved. Two fragments bound at the P stalk binding site and the third bound to helix D, a motif distinct from the P stalk binding site. All fragments bound RTA remote from the catalytic site and caused little change in catalytic site geometry. Two fragments uniquely bound at the hydrophobic pocket with affinity sufficient to inhibit the catalytic activity on eukaryotic ribosomes in the low micromolar range. The binding mode of these inhibitors mimicked the interaction of the P stalk peptide, establishing that small molecule inhibitors can inhibit RTA binding to the ribosome with the potential for therapeutic intervention.
Xiao-Ping Li; Rajesh K. Harijan; Jennifer Nielsen Kahn; Vern L. Schramm; Nilgun Ereken Tumer. Small Molecule Inhibitors Targeting the Interaction of Ricin Toxin A Subunit with Ribosomes. ACS Infectious Diseases 2020, 6, 1894 -1905.
AMA StyleXiao-Ping Li, Rajesh K. Harijan, Jennifer Nielsen Kahn, Vern L. Schramm, Nilgun Ereken Tumer. Small Molecule Inhibitors Targeting the Interaction of Ricin Toxin A Subunit with Ribosomes. ACS Infectious Diseases. 2020; 6 (7):1894-1905.
Chicago/Turabian StyleXiao-Ping Li; Rajesh K. Harijan; Jennifer Nielsen Kahn; Vern L. Schramm; Nilgun Ereken Tumer. 2020. "Small Molecule Inhibitors Targeting the Interaction of Ricin Toxin A Subunit with Ribosomes." ACS Infectious Diseases 6, no. 7: 1894-1905.
Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known
Nilgun E. Tumer. Introduction to the Toxins Special Issue “Ricin Toxins”. Toxins 2019, 12, 13 .
AMA StyleNilgun E. Tumer. Introduction to the Toxins Special Issue “Ricin Toxins”. Toxins. 2019; 12 (1):13.
Chicago/Turabian StyleNilgun E. Tumer. 2019. "Introduction to the Toxins Special Issue “Ricin Toxins”." Toxins 12, no. 1: 13.
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.
Michael Pierce; Diana Vengsarkar; John McLaughlin; Jennifer N. Kahn; Nilgun E. Tumer. Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress–induced HAC1 mRNA splicing on the ribosome. Journal of Biological Chemistry 2019, 294, 17848 -17862.
AMA StyleMichael Pierce, Diana Vengsarkar, John McLaughlin, Jennifer N. Kahn, Nilgun E. Tumer. Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress–induced HAC1 mRNA splicing on the ribosome. Journal of Biological Chemistry. 2019; 294 (47):17848-17862.
Chicago/Turabian StyleMichael Pierce; Diana Vengsarkar; John McLaughlin; Jennifer N. Kahn; Nilgun E. Tumer. 2019. "Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress–induced HAC1 mRNA splicing on the ribosome." Journal of Biological Chemistry 294, no. 47: 17848-17862.
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.
Yijun Zhou; Xiao-Ping Li; Jennifer Nielsen Kahn; John McLaughlin; Nilgun E. Tumer. Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin. Bioscience Reports 2019, 39, 1 .
AMA StyleYijun Zhou, Xiao-Ping Li, Jennifer Nielsen Kahn, John McLaughlin, Nilgun E. Tumer. Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin. Bioscience Reports. 2019; 39 (10):1.
Chicago/Turabian StyleYijun Zhou; Xiao-Ping Li; Jennifer Nielsen Kahn; John McLaughlin; Nilgun E. Tumer. 2019. "Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin." Bioscience Reports 39, no. 10: 1.
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.
Xiao-Ping Li; Jennifer N. Kahn; Nilgun E. Tumer. Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding. Toxins 2018, 10, 371 .
AMA StyleXiao-Ping Li, Jennifer N. Kahn, Nilgun E. Tumer. Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding. Toxins. 2018; 10 (9):371.
Chicago/Turabian StyleXiao-Ping Li; Jennifer N. Kahn; Nilgun E. Tumer. 2018. "Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding." Toxins 10, no. 9: 371.
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.
Yijun Zhou; Xiao-Ping Li; Jennifer N. Kahn; Nilgun E. Tumer. Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins. Toxins 2018, 10, 240 .
AMA StyleYijun Zhou, Xiao-Ping Li, Jennifer N. Kahn, Nilgun E. Tumer. Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins. Toxins. 2018; 10 (6):240.
Chicago/Turabian StyleYijun Zhou; Xiao-Ping Li; Jennifer N. Kahn; Nilgun E. Tumer. 2018. "Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins." Toxins 10, no. 6: 240.
The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.
Przemysław Grela; Xiao-Ping Li; Patrycja Horbowicz; Monika Dźwierzyńska; Marek Tchórzewski; Nilgun E. Tumer. Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus. Scientific Reports 2017, 7, 5608 .
AMA StylePrzemysław Grela, Xiao-Ping Li, Patrycja Horbowicz, Monika Dźwierzyńska, Marek Tchórzewski, Nilgun E. Tumer. Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus. Scientific Reports. 2017; 7 (1):5608.
Chicago/Turabian StylePrzemysław Grela; Xiao-Ping Li; Patrycja Horbowicz; Monika Dźwierzyńska; Marek Tchórzewski; Nilgun E. Tumer. 2017. "Human ribosomal P1-P2 heterodimer represents an optimal docking site for ricin A chain with a prominent role for P1 C-terminus." Scientific Reports 7, no. 1: 5608.
Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.
Xiao-Ping Li; Nilgun E. Tumer. Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins. Toxins 2017, 9, 133 .
AMA StyleXiao-Ping Li, Nilgun E. Tumer. Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins. Toxins. 2017; 9 (4):133.
Chicago/Turabian StyleXiao-Ping Li; Nilgun E. Tumer. 2017. "Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins." Toxins 9, no. 4: 133.
Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α–sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA.
Yijun Zhou; Xiao-Ping Li; Brian Y. Chen; Nilgun E. Tumer. Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk. Scientific Reports 2017, 7, 42912 .
AMA StyleYijun Zhou, Xiao-Ping Li, Brian Y. Chen, Nilgun E. Tumer. Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk. Scientific Reports. 2017; 7 (1):42912.
Chicago/Turabian StyleYijun Zhou; Xiao-Ping Li; Brian Y. Chen; Nilgun E. Tumer. 2017. "Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk." Scientific Reports 7, no. 1: 42912.
The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome.
Debaleena Basu; Jennifer N. Kahn; Xiao-Ping Li; Nilgun E. Tumer. Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome. Infection and Immunity 2016, 84, 3290 -3301.
AMA StyleDebaleena Basu, Jennifer N. Kahn, Xiao-Ping Li, Nilgun E. Tumer. Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome. Infection and Immunity. 2016; 84 (12):3290-3301.
Chicago/Turabian StyleDebaleena Basu; Jennifer N. Kahn; Xiao-Ping Li; Nilgun E. Tumer. 2016. "Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome." Infection and Immunity 84, no. 12: 3290-3301.
Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae . Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.
Debaleena Basu; Xiao-Ping Li; Jennifer N. Kahn; Kerrie L. May; Peter C. Kahn; Nilgun E. Tumer. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1. Infection and Immunity 2016, 84, 149 -161.
AMA StyleDebaleena Basu, Xiao-Ping Li, Jennifer N. Kahn, Kerrie L. May, Peter C. Kahn, Nilgun E. Tumer. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1. Infection and Immunity. 2016; 84 (1):149-161.
Chicago/Turabian StyleDebaleena Basu; Xiao-Ping Li; Jennifer N. Kahn; Kerrie L. May; Peter C. Kahn; Nilgun E. Tumer. 2016. "The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1." Infection and Immunity 84, no. 1: 149-161.
Note: In lieu of an abstract, this is an excerpt from the first page.Excerpt Plants express a variety of toxic proteins which are thought to have a role in defense against pathogens and insects.
Nilgun E. Tumer. Introduction to the Toxins Special Issue on Plant Toxins. Toxins 2015, 7, 4503 -4506.
AMA StyleNilgun E. Tumer. Introduction to the Toxins Special Issue on Plant Toxins. Toxins. 2015; 7 (11):4503-4506.
Chicago/Turabian StyleNilgun E. Tumer. 2015. "Introduction to the Toxins Special Issue on Plant Toxins." Toxins 7, no. 11: 4503-4506.
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.
John McLaughlin; Mohamed Anwar Bin-Umer; Thomas Widiez; Daniel Finn; Susan McCormick; Nilgun E. Tumer. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin. PLoS ONE 2015, 10, e0130204 .
AMA StyleJohn McLaughlin, Mohamed Anwar Bin-Umer, Thomas Widiez, Daniel Finn, Susan McCormick, Nilgun E. Tumer. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin. PLoS ONE. 2015; 10 (6):e0130204.
Chicago/Turabian StyleJohn McLaughlin; Mohamed Anwar Bin-Umer; Thomas Widiez; Daniel Finn; Susan McCormick; Nilgun E. Tumer. 2015. "A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin." PLoS ONE 10, no. 6: e0130204.
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.
Debaleena Basu; Nilgun E. Tumer. Do the A Subunits Contribute to the Differences in the Toxicity of Shiga Toxin 1 and Shiga Toxin 2? Toxins 2015, 7, 1467 -1485.
AMA StyleDebaleena Basu, Nilgun E. Tumer. Do the A Subunits Contribute to the Differences in the Toxicity of Shiga Toxin 1 and Shiga Toxin 2? Toxins. 2015; 7 (5):1467-1485.
Chicago/Turabian StyleDebaleena Basu; Nilgun E. Tumer. 2015. "Do the A Subunits Contribute to the Differences in the Toxicity of Shiga Toxin 1 and Shiga Toxin 2?" Toxins 7, no. 5: 1467-1485.
Pokeweed antiviral protein (PAP) is a 29 kDa type I ribosome inactivating protein (RIP) found in pokeweed plants. Pokeweed produces different forms of PAP. This review focuses on the spring form of PAP isolated from Phytolacca americana leaves. PAP exerts its cytotoxicity by removing a specific adenine from the α-sarcin/ricin loop of the large ribosomal RNA. Besides depurination of the rRNA, PAP has additional activities that contribute to its cytotoxicity. The mechanism of PAP cytotoxicity is summarized based on evidence from the analysis of transgenic plants and the yeast model system. PAP was initially found to be anti-viral when it was co-inoculated with plant viruses onto plants. Transgenic plants expressing PAP and non-toxic PAP mutants have displayed broad-spectrum resistance to both viral and fungal infection. The mechanism of PAP-induced disease resistance in transgenic plants is summarized.
Rong Di; Nilgun E. Tumer. Pokeweed Antiviral Protein: Its Cytotoxicity Mechanism and Applications in Plant Disease Resistance. Toxins 2015, 7, 755 -772.
AMA StyleRong Di, Nilgun E. Tumer. Pokeweed Antiviral Protein: Its Cytotoxicity Mechanism and Applications in Plant Disease Resistance. Toxins. 2015; 7 (3):755-772.
Chicago/Turabian StyleRong Di; Nilgun E. Tumer. 2015. "Pokeweed Antiviral Protein: Its Cytotoxicity Mechanism and Applications in Plant Disease Resistance." Toxins 7, no. 3: 755-772.
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.
Qing Yan; Xiao-Ping Li; Nilgun E. Tumer. Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in Saccharomyces cerevisiae. PLOS ONE 2014, 9, e113719 .
AMA StyleQing Yan, Xiao-Ping Li, Nilgun E. Tumer. Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in Saccharomyces cerevisiae. PLOS ONE. 2014; 9 (12):e113719.
Chicago/Turabian StyleQing Yan; Xiao-Ping Li; Nilgun E. Tumer. 2014. "Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in Saccharomyces cerevisiae." PLOS ONE 9, no. 12: e113719.
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.
Mohamed Anwar Bin-Umer; John McLaughlin; Matthew S. Butterly; Susan McCormick; Nilgun E. Tumer. Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes. Proceedings of the National Academy of Sciences 2014, 111, 11798 -11803.
AMA StyleMohamed Anwar Bin-Umer, John McLaughlin, Matthew S. Butterly, Susan McCormick, Nilgun E. Tumer. Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes. Proceedings of the National Academy of Sciences. 2014; 111 (32):11798-11803.
Chicago/Turabian StyleMohamed Anwar Bin-Umer; John McLaughlin; Matthew S. Butterly; Susan McCormick; Nilgun E. Tumer. 2014. "Elimination of damaged mitochondria through mitophagy reduces mitochondrial oxidative stress and increases tolerance to trichothecenes." Proceedings of the National Academy of Sciences 111, no. 32: 11798-11803.
Wild-type yeast ribosomes and ribosomes bearing only the P1B–P2A dimer bound to the ricin A chain better and were more susceptible to depurination and toxicity than ribosomes bearing only the P1A–P2B dimer, indicating that the two stalk dimers differ in their function.
Przemysław Grela; Xiao-Ping Li; Marek Tchórzewski; Nilgun E. Tumer. Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain. Biochemical Journal 2014, 460, 59 -69.
AMA StylePrzemysław Grela, Xiao-Ping Li, Marek Tchórzewski, Nilgun E. Tumer. Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain. Biochemical Journal. 2014; 460 (1):59-69.
Chicago/Turabian StylePrzemysław Grela; Xiao-Ping Li; Marek Tchórzewski; Nilgun E. Tumer. 2014. "Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain." Biochemical Journal 460, no. 1: 59-69.