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Stephen H. Leppla - One of the best experts on this subject based on the ideXlab platform.
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inhibitory effects of a reengineered Anthrax Toxin on canine and human osteosarcoma cells
Toxins, 2020Co-Authors: Jonathan M Fonseca, Stephen H. Leppla, Shihui Liu, Thomas H Bugge, Adriana Tomoko Nishiya, Marcia Kazumi Nagamine, Ivone Izabel Mackowiak Da Fonseca, Jerrold M Ward, Cristina De Oliveira Massoco, M L DagliAbstract:Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis Toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered Toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered Anthrax Toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the Toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered Anthrax Toxin on OSA cells; this reengineered Toxin could be further tested as a new therapy for OSA.
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Bismaleimide cross-linked Anthrax Toxin forms functional octamers with high specificity in tumor targeting.
Protein science : a publication of the Protein Society, 2019Co-Authors: Elyse S. Fischer, Shihui Liu, Thomas H Bugge, Rasem J Fattah, Warren A. Campbell, Rodolfo Ghirlando, Stephen H. LepplaAbstract:In recent years, Anthrax Toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional Toxins upon complementary oligomerization. Here, we have created a dual-protease requiring PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross-linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the Anthrax Toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, Anthrax Toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross-linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of Anthrax Toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials.
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Effect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the Anthrax Toxin channel activity.
Biochimica et biophysica acta. Biomembranes, 2018Co-Authors: Nnanya Kalu, Stephen H. Leppla, Yoav Atsmon-raz, Sanaz Momben Abolfath, Laura Lucas, Clare Kenney, D. Peter Tieleman, Ekaterina M. NestorovichAbstract:Abstract Anthrax Toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the Toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the Toxin's enzymatic components into the cytosol. Membrane lipid composition varies at different stages of Anthrax Toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(monoacylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the Anthrax Toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The Anthrax Toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.
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Tumor Targeting and Drug Delivery by Anthrax Toxin.
Toxins, 2016Co-Authors: Christopher Bachran, Stephen H. LepplaAbstract:Anthrax Toxin is a potent tripartite protein Toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease Anthrax. The receptor-binding component of the Toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the Toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting Anthrax Toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of Anthrax Toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different Toxin variants that require activation by two different tumor-associated proteases for increased specificity of Toxin activation. The Anthrax Toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.
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key tissue targets responsible for Anthrax Toxin induced lethality
Nature, 2013Co-Authors: Yi Zhang, Devorah Crown, Mahtab Moayeri, Rasem J Fattah, Alexander N Wein, Zu Xi Yu, Toren Finkel, Stephen H. LepplaAbstract:Cell-type-specific Anthrax Toxin receptor CMG2-null mice are generated and used to show that the Bacillus anthracis Toxins lethal Toxin (LT) and oedema Toxin (ET) target distinct cell types; in contrast to previous suggestions, it is shown that endothelial cells are not key targets for either Toxin and instead LT targets cardiomyocytes and vascular smooth muscle cells whereas ET targets hepatocytes.
Shihui Liu - One of the best experts on this subject based on the ideXlab platform.
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inhibitory effects of a reengineered Anthrax Toxin on canine and human osteosarcoma cells
Toxins, 2020Co-Authors: Jonathan M Fonseca, Stephen H. Leppla, Shihui Liu, Thomas H Bugge, Adriana Tomoko Nishiya, Marcia Kazumi Nagamine, Ivone Izabel Mackowiak Da Fonseca, Jerrold M Ward, Cristina De Oliveira Massoco, M L DagliAbstract:Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis Toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered Toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered Anthrax Toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the Toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered Anthrax Toxin on OSA cells; this reengineered Toxin could be further tested as a new therapy for OSA.
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Bismaleimide cross-linked Anthrax Toxin forms functional octamers with high specificity in tumor targeting.
Protein science : a publication of the Protein Society, 2019Co-Authors: Elyse S. Fischer, Shihui Liu, Thomas H Bugge, Rasem J Fattah, Warren A. Campbell, Rodolfo Ghirlando, Stephen H. LepplaAbstract:In recent years, Anthrax Toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic, shown to kill tumors in animal models. This has been achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA), which are upregulated in tumor microenvironments. These therapeutics consist of intercomplementing PA variants, which are individually nontoxic, but form functional Toxins upon complementary oligomerization. Here, we have created a dual-protease requiring PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of the CLPA agents have in vitro toxicities equal to those of previous intercomplementing agents, while the third CLPA agent had compromised in vitro cleavage and was significantly less cytotoxic. We hypothesize this difference was due to steric hindrance caused by cross-linking two PA monomers in close proximity to the PA cleavage site. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the Anthrax Toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. HIGHLIGHT: Previously, Anthrax Toxin has been reengineered to act as a highly specific antiangiogenic cancer therapeutic. Here, we present a version, which utilizes bismaleimide cross-linked protective antigen (PA) rather than intercomplementing PA variants. This advances the development of Anthrax Toxin as a practical cancer therapeutic as it reduces the components of the drug delivery system to two, which may lower the cost and simplify testing in clinical trials.
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Key tissue targets responsible for Anthrax-Toxin-induced lethality
Nature, 2013Co-Authors: Shihui Liu, Devorah Crown, Mahtab Moayeri, Yi Zhang, Rasem J Fattah, Alexander N Wein, Toren Finkel, Jie Liu, Stephen H. LepplaAbstract:Bacillus anthracis, the causative agent of Anthrax disease, is lethal owing to the actions of two exoToxins: Anthrax lethal Toxin (LT) and oedema Toxin (ET). The key tissue targets responsible for the lethal effects of these Toxins are unknown. Here we generated cell-type-specific Anthrax Toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the Toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either Toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems. Cell-type-specific Anthrax Toxin receptor CMG2-null mice are generated and used to show that the Bacillus anthracis Toxins lethal Toxin (LT) and oedema Toxin (ET) target distinct cell types; in contrast to previous suggestions, it is shown that endothelial cells are not key targets for either Toxin and instead LT targets cardiomyocytes and vascular smooth muscle cells whereas ET targets hepatocytes. Bacillus anthracis produces two Toxins — Anthrax lethal Toxin and oedema Toxin — which are targeted to tissues by captor-associated protective antigen. The two Toxins have essential but little understood roles in pathogenesis. Here, Shihui Liu and colleagues generate mice lacking cell-type-specific Anthrax Toxin receptor capillary morphogenesis protein-2 (CMG2) and use them to show that the two Toxins target distinct cell types. Contrary to previous suggestions, endothelial cells are not key targets for either Toxin. Rather, lethal Toxin targets cardiomyocytes and vascular smooth muscle cells, and oedema Toxin targets hepatocytes. Recognition that the Anthrax Toxins are specifically targeting the cardiovascular system and liver may suggest supportive therapies that would limit tissue damage and increase survival in human Anthrax infections.
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The Receptors that Mediate the Direct Lethality of Anthrax Toxin
Toxins, 2012Co-Authors: Shihui Liu, Yi Zhang, Benjamin Hoover, Stephen H. LepplaAbstract:Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized Anthrax Toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for Anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third Anthrax Toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as Anthrax Toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of Anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exoToxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8−/− and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2−/− mice were completely resistant to this dose, confirming that CMG2 is the major Anthrax Toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2−/−/TEM8−/− mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2−/−/TEM8−/− mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor Anthrax Toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.
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Anthrax Toxin targeting of myeloid cells through the cmg2 receptor is essential for establishment of bacillus anthracis infections in mice
Cell Host & Microbe, 2010Co-Authors: Shihui Liu, Devorah Crown, Mahtab Moayeri, Sharmina Millerrandolph, Inka Sastalla, Shu Okugawa, Stephen H. LepplaAbstract:Bacillus anthracis kills through a combination of bacterial infection and toxemia. Anthrax Toxin working via the CMG2 receptor mediates lethality late in infection, but its roles early in infection remain unclear. We generated myeloid-lineage specific CMG2-deficient mice to examine the roles of macrophages, neutrophils, and other myeloid cells in Anthrax pathogenesis. Macrophages and neutrophils isolated from these mice were resistant to Anthrax Toxin. However, the myeloid-specific CMG2-deficient mice remained fully sensitive to both Anthrax lethal and edema Toxins, demonstrating that targeting of myeloid cells is not responsible for Anthrax Toxin-induced lethality. Surprisingly, the myeloid-specific CMG2-deficient mice were completely resistant to B. anthracis infection. Neutrophil depletion experiments suggest that B. anthracis relies on Anthrax Toxin secretion to evade the scavenging functions of neutrophils to successfully establish infection. This work demonstrates that Anthrax Toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to Anthrax infection.
John A. T. Young - One of the best experts on this subject based on the ideXlab platform.
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Receptors of Anthrax Toxin and cell entry
Molecular aspects of medicine, 2009Co-Authors: Gisou Van Der Goot, John A. T. YoungAbstract:Anthrax Toxin-receptor interactions are critical for Toxin delivery to the host cell cytoplasm. This review summarizes what is known about the molecular details of the protective antigen (PA) Toxin subunit interaction with either the ANTXR1 and ANTXR2 cellular receptors, and how receptor-type can dictate the low pH threshold of PA pore formation. The roles played by cellular factors in regulating the endocytosis of Toxin-receptor complexes is also discussed.
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Evidence against a human cell-specific role for LRP6 in Anthrax Toxin entry.
PloS one, 2008Co-Authors: Patricia L. Ryan, John A. T. YoungAbstract:The role of the cellular protein LRP6 in Anthrax Toxin entry is controversial. Previous studies showed that LRP6 was important for efficient intoxication of human M2182 prostate carcinoma cells but other studies performed with cells from gene-knockout mice demonstrated no role for either LRP6 or the related LRP5 protein in Anthrax Toxin entry. One possible explanation for this discrepancy is that LRP6 may be important for Anthrax Toxin entry into human, but not mouse, cells. To test this idea we have investigated the effect of knocking down LRP6 or LRP5 expression with siRNAs in human HeLa cells. We show here that efficient knockdown of either LRP6, LRP5, or both proteins has no influence on the kinetics of Anthrax lethal Toxin entry or MEK1 substrate cleavage in these cells. These data argue against a human-specific role for LRP6 in Anthrax Toxin entry and suggest instead that involvement of this protein may be restricted to certain cell types independently of their species of origin.
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Anthrax Toxin receptor 2-dependent lethal Toxin killing in vivo.
PLoS pathogens, 2006Co-Authors: Heather M. Scobie, Darran J Wigelsworth, John M. Marlett, Diane Thomas, G. Jonah A. Rainey, D. Borden Lacy, Marianne Manchester, R. John Collier, John A. T. YoungAbstract:Anthrax Toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of Anthrax Toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by Anthrax lethal Toxin, providing strong evidence for the physiological importance of ANTXR2 in Anthrax disease pathogenesis.
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Interactions between Anthrax Toxin receptors and protective antigen.
Current opinion in microbiology, 2005Co-Authors: Heather M. Scobie, John A. T. YoungAbstract:Since the Anthrax mail attacks of 2001, much has been learned about the interactions between Anthrax Toxin and its receptors. Two distinct cellular receptors for Anthrax Toxin have been identified and are designated capillary morphogenesis protein 2 (CMG2) and Anthrax Toxin receptor/tumor endothelial marker 8 (ATR/TEM8). The molecular details of the Toxin-receptor interactions have been revealed through crystallographic, biochemical and genetic studies. In addition, a novel pathway by which Anthrax Toxin enters cells is starting to be uncovered.
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Binding of Anthrax Toxin to its receptor is similar to α integrin-ligand interactions
The Journal of biological chemistry, 2003Co-Authors: Kenneth A. Bradley, Jeremy Mogridge, G. Jonah, A. Rainey, Sarah Batty, John A. T. YoungAbstract:The secreted protein Toxin produced by Bacillus anthracis contributes to virulence of this pathogen and can cause many of the symptoms seen during an Anthrax infection, including shock and sudden death. The cell-binding component of Anthrax Toxin, protective antigen, mediates entry of the Toxin into cells by first binding directly to the extracellular integrin-like inserted (I) domain of the cellular Anthrax Toxin receptor, ATR. Here we report that this interaction requires an intact metal ion-dependent adhesion site (MIDAS) in the receptor as well as the presence of specific divalent cations. Also, we demonstrate that the Toxin-receptor interaction is critically dependent on the Asp-683 carboxylate group of protective antigen, which projects from the receptor binding surface. We propose that this carboxylate group completes the coordination of the MIDAS metal of ATR, mimicking integrin-ligand interactions.
Laurence Abrami - One of the best experts on this subject based on the ideXlab platform.
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Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors
PLoS pathogens, 2010Co-Authors: Laurence Abrami, Béatrice Kunz, Mirko Bischofberger, Romain Groux, F. Gisou Van Der GootAbstract:The Anthrax Toxin is a tripartite Toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the Toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a beta-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the Anthrax Toxin.
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Anthrax Toxin triggers the activation of src-like kinases to mediate its own uptake
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Laurence Abrami, Béatrice Kunz, F. Gisou Van Der GootAbstract:AB-type Toxins, like other bacterial Toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The Anthrax Toxin, however, times its own uptake, suggesting that Toxin binding triggers specific signaling events. Here we show that the Anthrax Toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient Toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon Toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the Anthrax Toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.
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Functional interactions between Anthrax Toxin receptors and the WNT signalling protein LRP6.
Cellular microbiology, 2008Co-Authors: Laurence Abrami, Béatrice Kunz, Julie Deuquet, Anna Bafico, Gary Davidson, F. Gisou Van Der GootAbstract:To exert its activity, Anthrax Toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the Anthrax Toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for Toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in Anthrax Toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in Anthrax Toxin endocytosis, but is not essential. We indeed found that Toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed Toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR-LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.
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receptor palmitoylation and ubiquitination regulate Anthrax Toxin endocytosis
Journal of Cell Biology, 2006Co-Authors: Laurence Abrami, Stephen H. Leppla, Gisou F Van Der GootAbstract:The Anthrax Toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to Anthrax Toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.
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Anthrax Toxin triggers endocytosis of its receptor via a lipid raft mediated clathrin dependent process
Journal of Cell Biology, 2003Co-Authors: Laurence Abrami, Pierre Cosson, Stephen H. Leppla, Gisou F Van Der GootAbstract:The protective antigen (PA) of the Anthrax Toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the Anthrax Toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against Anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of Anthrax Toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal Anthrax Toxin receptor.
F. Gisou Van Der Goot - One of the best experts on this subject based on the ideXlab platform.
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Endocytosis of the Anthrax Toxin Is Mediated by Clathrin, Actin and Unconventional Adaptors
PLoS pathogens, 2010Co-Authors: Laurence Abrami, Béatrice Kunz, Mirko Bischofberger, Romain Groux, F. Gisou Van Der GootAbstract:The Anthrax Toxin is a tripartite Toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the Toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a beta-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the Anthrax Toxin.
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Anthrax Toxin triggers the activation of src-like kinases to mediate its own uptake
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Laurence Abrami, Béatrice Kunz, F. Gisou Van Der GootAbstract:AB-type Toxins, like other bacterial Toxins, are notably opportunistic molecules. They rely on target cell receptors to reach the appropriate location within the target cell where translocation of their enzymatic subunits occurs. The Anthrax Toxin, however, times its own uptake, suggesting that Toxin binding triggers specific signaling events. Here we show that the Anthrax Toxin triggers tyrosine phosphorylation of its own receptors, capillary morphogenesis gene 2 and tumor endothelial marker 8, which are not endowed with intrinsic kinase activity. This is required for efficient Toxin uptake because endocytosis of the mutant receptor lacking the cytoplasmic tyrosine residues is strongly delayed. Phosphorylation of the receptors was dependent on src-like kinases, which where activated upon Toxin binding. Importantly, src-dependent phosphorylation of the receptor was required for its subsequent ubiquitination, which in turn was required for clathrin-mediated endocytosis. Consistently, we found that uptake of the Anthrax Toxin and processing of the lethal factor substrate MEK1 are inhibited by silencing of src and fyn, as well as in src and fyn knockout cells.
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Functional interactions between Anthrax Toxin receptors and the WNT signalling protein LRP6.
Cellular microbiology, 2008Co-Authors: Laurence Abrami, Béatrice Kunz, Julie Deuquet, Anna Bafico, Gary Davidson, F. Gisou Van Der GootAbstract:To exert its activity, Anthrax Toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the Anthrax Toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for Toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in Anthrax Toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in Anthrax Toxin endocytosis, but is not essential. We indeed found that Toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed Toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR-LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.
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Anthrax Toxin triggers endocytosis of its receptor via a lipid raft–mediated clathrin-dependent process
The Journal of cell biology, 2003Co-Authors: Laurence Abrami, Pierre Cosson, Stephen H. Leppla, Shihui Liu, F. Gisou Van Der GootAbstract:The protective antigen (PA) of the Anthrax Toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the Anthrax Toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against Anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of Anthrax Toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal Anthrax Toxin receptor.